nicotinamide adeninedinucleotide biosynthesis …84 fosterandmoat n..,conh2 o _o_p_o_co + |hh o ho...

MICROBIOLOGICAL REVIEWS, Ma. 1980, p. 83-105 Vol. 44, No. 10146-0749/80/01-0083/23$02.00/0

Nicotinamide Adenine Dinucleotide Biosynthesis and PyridineNucleotide Cycle Metabolism in Microbial Systems

JOHN W. FOSTER* AND ALBERT G. MOATDepartment ofMicrobiology, Marshall University School ofMedicine, Huntington, West Virginia 25701

INTRODUCTION ...... ......... .......... .................. 83BIOSYNTHESIS OF NICOTINAMIDE ADENINE DINUCLEOTIDE 83Anaerobic De Novo Pathways .. ...... .... .... ... 84Dihydroxyacetone phosphate-aspartate pathway 84Formate-aspartate pathway .............................................. 87

Aerobic Tryptophan Catabolic Pathway 87Genetics of Anaerobic Nicotinamide Adenine Dinucleotide Biosynthesis .... 89Regulation of De Novo Biosynthesis of Nicotinamide Adenine Dinucleotide 91

PYRIDINE NUCLEOTIDE CYCLE METABOLISM ...................... 93Biochemistry .............................. 93Pyridine Nucleotide Cycle Genetics.95Regulation.96Organisms of Special Interest 98Haenwphilus ...................9.8......................98Mycobacterium ................ .... 98Clostridium butylicum ....................... 98Azotobacter vinelandii ......... .................. ............ ......... 99Lactobacillus and Leuconostoc 99

EVOLUTIONARY ASPECTS 99Evolution of Biosynthetic Pathways 99Evolution of the Pyridine Nucleotide Cycle........ 100

SUMMARY.100LITERATURE CITED ..... ............................ ... ... ... 102

INTRODUCTIONNicotinamide adenine dinucleotide (NAD)

and NAD-phosphate (NADP) are compounds ofimmeasurable importance in cellular metabo-lism. They function in numerous anabolic andcatabolic reactions and are widely distributedthroughout biological systems. The structures ofthese compounds are presented in Fig. 1 (113).NAD and NADP are known to participate inover 300 enzymatically catalyzed oxidation-re-duction reactions. In addition, a number of re-actions have been discovered in which NADserves as a substrate. For example, certain pro-caryotes, such as Escherichia coli, utilize NADas a substrate for deoxyribonucleic acid ligase,an essential for deoxyribonucleic acid synthesis,repair, and recombination (68, 80). NAD alsoserves as a substrate in reactions that producepoly-adenosine 5'-diphosphate-ribose (47, 104).Adenosine 5'-diphosphate ribosylation is prov-ing to be of great importance to both eucaryoticand procaryotic cells (20, 21, 44, 47). Reducedpyridine nucleotide coenzymes also play an im-portant role in the regulation of amphibolicpathways, such as the citric acid cycle and theoxidative pentose pathway (91). Thus, there is agrowing awareness of the extent to which cellsare dependent upon NAD and an increased em-

phasis on the need for more extensive researchinto the synthesis, recycling, and regulation ofNAD metabolism.No comprehensive review on NAD metabo-

lism has been published since the review byChaykin in 1967 (16). Meanwhile, a considerableamount of work has been published on thissubject. The purpose of this article, then, is tosummarize the most recent developments re-garding the biochemistry ofNAD metabolism aswell as the regulation and genetics of this sys-tem. Emphasis will be placed on the E. coli-Salmonella typhimurium systems, since theyare understood best. For the reader's conveni-ence, Tables 1 and 2 present a summary of theenzymes considered in this review.

BIOSYNTHESIS OF NICOTINAMIDEADENINE DINUCLEOTIDE

Extensive research on the biosynthesis ofNAD has been undertaken in mammalian andlower eucaryotic systems. Similar studies in pro-caryotes have not been pursued as widely. Thisis surprising in view of the potential significanceassociated with the evolutionary development ofprocaryotic systems for NAD biosynthesis. Areview of the literature reveals two main biosyn-thetic pathways to NAD. One pathway involves

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84 FOSTER AND MOAT

N..,CONH2

O_O_P_O_Co +

|HHo HO NH20H

-0-p- H

HO OHtLO PO3

FIG. 1. Molecular structures of NAD and NADP.The arrow indicates the point at which the thirdphosphate group is added by NAD kinase to formNADP.

the aerobic degradation of tryptophan by mam-malian cells and a number of lower eucaryotes.Another pathway, found predominantly in pro-caryotes, is anaerobic and utilizes low-molecu-lar-weight precursors for the synthesis of thepyridine ring structure of NAD. Both pathwayslead to the formation of quinolinic acid (QA).Subsequent conversion of QA to NAD occursvia a pathway common to all organisms thathave been examined to date.

Anaerobic De Novo PathwaysRelatively few procaryotic species are capable

of using tryptophan for the formation of thepyridine ring of NAD. Xanthomonas pruni canconvert tryptophan to NAD (16, 117), and theenzymes in the pathway have been well docu-mented. Some members of the actinomycetegroup also appear to utilize tryptophan for NADbiosynthesis (70). In view of the importance ofNAD and NADP in procaryotic metabolism, itis surprising that investigations as to the routeof synthesis of these essential cofactors havebeen so late in developing.Dihydroxyacetone phosphate-aspartate

pathway. The first attempt to elucidate a pro-caryotic pathway to NAD was reported by Or-tega and Brown in 1960 (81). They implicatedglycerol and a dicarboxylic acid as precursors inthe synthesis of the pyridine ring of NAD by E.coli. Subsequently, Andreoli and his co-workersdemonstrated that QA was a key intermediatein this de novo pathway (4). QA is now recog-nized as a precursor involved in all known bio-

synthetic pathways to NAD (Fig. 2). Subsequentwork by Chandler et al. (15) established L-as-partic acid as the dicarboxylic acid precursor.Suzuki et al., in 1973, established that the three-carbon precursor is dihydroxyacetone phosphate(DHAP) and not glycerol (107). L-Aspartic acidand DHAP undergo a condensation reaction toform an intermediate which is cyclized to formthe pyridine ring of QA. Labeling studies haveshown that C-3 of DHAP condenses with C-3 ofaspartate (116). Similar studies with Mycobac-terium tuberculosis have unequivocally estab-lished that the aspartate nitrogen and carbonare incorporated intact into the pyridine ring(42). The condensation between aspartate andDHAP is a two-enzyme step, collectively termedthe QA synthetase system, for which an inter-mediate has not yet been isolated in pure form(Fig. 3).Mutants defective in this de novo pathway

have been isolated from E. coli and S. typhi-murium, and the relevant genes were designatednadA, nadB, nadC, and nadR (see Genetics ofAnaerobic Nicotinamide Adenine DinucleotideBiosynthesis). Chen and Tritz (17) have isolatedfrom nadC mutants of E. coli a metabolite thatis capable of supporting the growth of bothnadA and nadB mutants but is incapable ofsupporting the growth of nadC mutants (QAphosphoribosyltransferase [QAPRTase] defi-cient). This metabolite has not been character-ized completely, but it is doubtful that it is thehypothesized intermediate represented as [X] inFig. 3 due to the lack of differentiation in thesupport of growth of nadA and nadB mutants.Tritz theorizes that this compound is either theimmediate precursor of QA which cyclizes non-enzymatically or an intermediate in an alternatepathway leading to QA. Kerr and Tritz (61) havealso shown that some, but not all, Nad- mutantsin each class (nadA, nadB, nadC, and nadR)can grow in a vitamin-free Casamino Acids me-dium without nicotinic acid (NA). The remotepossibility exists that these "dichotomistic" mu-tants are capable of utilizing an alternate path-way when stimulated by certain amino acids.The compound isolated by Chen and Tritz (17)could be an intermediate in this alternate path-way.More recently, these investigators have pre-

sented evidence which suggests that up to sixintermediates may be involved in the formationofQA from L-aspartate and DHAP. Experimentswere conducted in which the QA synthetasesystem was provided with either [14C]aspartateor [14C]fructose-1,6-diphosphate (as a source ofDHAP) and the reaction mixtures were chro-matographed. Autoradiograms of each chromat-ogram revealed a common spot, providing evi-

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NAD BIOSYNTHESIS AND PNC METABOLISM

TABLE 1. Enzymes involved in anaerobic NAD biosynthesis and PNC metabolismaEnzyme nomenclatureb Map position

Name|EC no. | Reaction Alternate name loGenetic SName ECno.'uru E. coli

QA synthetase

QAPRTase

NAMNadenylyltransferase

NAD synthetase

NAD (NADH)pyrophosphatase

2.4.2.19

2.7.7.18

6.3.5.1

3.6.1.22

NAD glycohydrolase 13.2.2.5

L-Asp + DHAP + FAD- o QA +H3P04 + 2H20 + FADH2

Mg2+QA + PRPP- NAMN + PPi +C02

Mg2+NAMN + ATP- . deNAD + PPiMg2+

NMN + ATP- NAD + PPi

deNAD + ATP + glutamine (NH3)Mg2+M- NAD + PPi + AMP

NAD+H20 m- AMP + NMN

NADP + H20 m- , 2',5'-AMP +NMN

Mg2+NAD + H20 - I NAm + ADP-ribose

NMN glycohydrolase 3.2.2.00 NMN + H20 - NAm + ribose-5-phosphate

NMNamidohydrolase

NAmamidohydrolase

NAPRTase

NAD kinase

NADP phosphatase

DNA ligase

NADH kinase

NAmPRTase

NMNadenylyltransferase

3.5.1.00 NMN + H20 -* NAMN + NH3

3.5.1.19 NAm + H20 - NA + NH3

2.4.2.11Mg2+

NA + PRPP + ATP - NAMN +PPi + ADP + P

2.7.1.23 NAD + ATPMg2+ NADP + ADP

3.1.2.00

6.5.1.2

2.4.2.12

2.7.7.1

Mg2+NADP + H20 -. NAD + H3P04

NAD + nicked DNA -e , NMN +AMP + DNA

NADH + ATP -e , NADPH +ADP

deNADpyrophosphorylase

NAD nucleosidase

NMN deamidase

NAm deamidase

NAMNpyrophosphorylase

NAm+PRPP+ATP-NMN+PP NMN+ ADP + Pi pyrophosphorylase

NMN + PRPP + ATP - NAD + PPi NAD+ ADP + Pi pyrophosphorylase

nadAnadB

nadC

Absent

pncC

pncA

pncB

lg

Absent

1758

3

27

25

1655

2

39

51

a Abbreviations: PRPP, phosphoribosyl pyrophosphate; PPi, inorganic pyrophosphate; AMP, adenosine 5'-monophosphate;ADP, adenosine 5'-diphosphate; Pi, inorganic phosphate; Asp, aspartate; DHAP, dihydroxyacetone phosphate; QA, quinolinicacid; NA, nicotinic acid; NAm, nicotinamide; NAMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide;deNAD, desamido-nicotinamide adenine dinucleotide or nicotinic acid adenine dinucleotide; NAD, nicotinamide adeninedinucleotide; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotidephosphate; QAPRTase, quinolinic acid phosphoribosyltransferase; NAPRTase, nicotinic acid phosphoribosyltransferase; DNA,deoxyribonucleic acid.

'Recommended names and Enzyme Commission (EC) numbers are taken from reference 53a.

dence for the formation of a compound (or com- tion with partially purified material revealedpounds) labeled by both aspartate and fructose- that the reaction mixture contained six labeled1,6-diphosphate (18). Subsequent experimenta- spots when ["4C]aspartate was used, whereas

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86 FOSTER AND MOAT

TABLE 2. Enzymes involved in the aerobic tryptophan catabolic pathway to NADEnzyme nomenclaturea

Alternate name(s) ReactionName EC no.

Tryptophan 2,3- 1.13.11.11 Tryptophan pyrrolase, L-Tryptophan + 02 -dioxygenase tryptophan oxygenase L-formylkynurenine

Kynurenine 3.5.1.9 Formylase, N-Formyl-L-kynurenine + H20formamidase formylkynureninase formate + L-kynurenine

Kynurenine 3- 1.14.13.9 Kynurenine 3-hydroxylase L-Kynurenine + NADPH + 02monooxygenase 3-hydroxy-L-kynurenine + NADP+ +

H203-Hydroxykynureninase 3-Hydroxykynurenine

3-hydroxyanthranilate + alanine3-Hydroxyanthranilate 1.13.11.6 3-Hydroxyanthranilate 3-Hydroxyanthranilate + 02-s 2-amino-

3,4-dioxygenase oxygenase 3-carboxymuconate semialdehydea Recommended names and Enzyme Commission (EC) numbers are taken from reference 53a.

AEROBICA-

ANAEROBIC

TRYPTOPHAN DHAP + ASPARTATE FORMATE + ASPARTATE

QxIA -

aCOOH

NA OOHOA

t%NFCOOH 3COOH

N A _IAM

t (i) / ~NAN \ i3

CYONH2 COOH

K.Pp -P-P-RNMN deNAD

t3CONHI tONH,

N (f AdNAm --

NAD

FIG. 2. Pathways ofNAD biosynthesis and the four-, five-, and six-step pyridine nucleotide cycles. X refersto the unknown intermediate(s) in the anaerobic biosynthetic pathways to QA. Abbreviations: R, ribose; P,phosphate; Ad, adenine.

four spots were observed when ["C]fructose-1,6-diphosphate was used. Interestingly, the fourlabeled spots derived from ["4C]fructose-1,6-di-phosphate were identical to four of the labeledspots derived from ["4C]aspartate, indicatingthat two modifications of aspartate may occur

before it condenses with DHAP. Furthermore,syntheses of all six compounds were inhibited bythe addition of NAD to the reaction mixture(18).Gholson and his co-workers have extensively

examined the enzymes involved with QA biosyn-

COOH

NA

Ar

,~.CONH2

NAm

(CONHt

NMN

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thesis in E. coli. They have confirmed the re-quirement for two proteins: (i) the "A" protein,coded for by the nadA gene, and (ii) the "B"protein, the product of the nadB gene (107).They have separated the nadB gene productfrom the nadA gene product via sodium citratefractionation and have purified it by using var-ious column chromatographic techniques (41).From the ease of separation of the two proteincomponents, it was concluded that QA synthe-tase, if it exists as a complex, is not a tightlybound complex. Furthermore, a number of find-ings were presented which suggested that thenadB protein converts aspartate to an interme-diate capable of undergoing condensation withDHAP catalyzed by the nadA protein. The Bprotein requires flavin adenine dinucleotide foractivity. Therefore, the intermediate formedcould be an unstable dehydrogenation productof aspartate which might be difficult to isolateand identify.Recently there has been some question as to

whether or not the DHAP-aspartate pathway istruly an anaerobic pathway. S. Nasu, F. D.Wicks, and R. K. Gholson (Fed. Proc. 38:644,1979) have shown that the nadB component ofthe QA synthetase system exhibits a strict re-quirement for 02 when assayed in vitro. Thesuggestion has been made that under anaerobicconditions E. coli, and presumably S. typhimu-rium, would switch to a true anaerobic pathway,such as the N-formylaspartate pathway (see be-low). If this were true, however, one would ex-pect nadB mutants to lose their requirement forNA under anaerobic conditions, since an alter-nate pathway could be used. This has not beendemonstrated. Another possibility, however, isthat under anaerobic conditions the nadB geneproduct utilizes an electron acceptor other thanoxygen. This hypothesis predicts that a nadB

mutant will retain its Nad- phenotype underanaerobic conditions. Recently, 10 nadB mu-tants and several nadA mutants of S. typhimu-rium were tested for the Nad- phenotype underanaerobic conditions. All retained their require-ment for NA (J. W. Foster, unpublished data).Thus, for the purpose of this review we willpresent the DHAP-aspartate pathway as onewhich can function anaerobically.Formate-aspartate pathway. The gram-

positive anaerobe Clostridium butylicum utilizesa de novo pathway distinct from that describedabove. Research with this species has revealedthe precursors ofQA to be L-aspartate, formate,and acetyl coenzyme A (54, 96, 97). Aspartateand formate are condensed to form N-formylas-partate, which subsequently is condensed withacetyl coenzyme A to form QA (Fig. 4). Nofurther investigations have been performed todetermine the nature of the intermediate(s) inthe C. butylicum pathway.

Aerobic Tryptophan Catabolic PathwayThe role of tryptophan as a precursor in eu-

caryotic NAD biosynthesis was first suggestedby nutritional studies in which humans strickenwith pellagra, an NA deficiency disease, re-covered from this illness after the addition oftryptophan or niacin (NA) to their diets (66).Other studies established tryptophan as a pre-cursor of NAD in many animal and plant sys-tems (23).

In 1952, Yanofsky and his colleagues used themold Neurospora to elucidate some of the inter-mediate reactions in the synthesis of NAD fromtryptophan (84). The enzymatic steps were soonclarified through the use of radioisotope tracerexperiments and by the isolation of the variousintermediates involved. Figure 5 outlines thedegradation of tryptophan and subsequent syn-

HICO®§) COOH QA synthetase

HCO COOH A-§O=C HCH noB nadA no'N,OO s''c CO

H2COH CH FAD FADH2 pi Hi2c ';kN-COOHPR NHpN COOH

DHAP ASPARTATE QUINOLINATE NAMNFIG. 3. DHAP-L -aspartate pathway for QA biosynthesis. The genetic loci involved are designated nadA,

nadB, and nadC. X refers to the unknown intermediate(s) involved in the biosynthesis of QA. Abbreviations:®, phosphate; FAD, flavin adenine dinucleotide; FADH2, reduced FAD; P, inorganic phosphate; PRPP,

phosphoribosylpyrophosphate, R, ribose.

COOH COOH COOH/COOH C/ CH3CO-CoACH2 CM2 illHCOOH + I X

CH HCO CHHN/ACOOH\/\N OH

H2N COOH HN COOHFORMATE ASPARTATE N-FORMYLASPARTATE QUINOLINATE

FIG. 4. Formate-aspartate pathways for QA biosynthesis. CoA, Coenzyme A.

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88 FOSTER AND MOAT

1Fe~NlCOCH2CHCOOH 2~-NHCHO -CO

0 FORMYLKYNURENINE CO

rir-iI-CH2CHCOOH ~s%fNyCOCH2CHCOOHNH2~ ~ ~ ~ N.)NH2NH

H KYNURENI NETRYPTOPHAN N

02,NADPH 3

1 OCH2C H COOH

OH3-HYDROXYKYNURENINE

PAL -ALANINECOOH o s1COOH 402 r

0"H2NH C OOH NAPHNH2

6 /2-ACROLEYL-3 5 OHAMINO FUMARATE 3-HYDROXYANTHRANILATE

(COOH

COOHQUINOLINATE

PRPP |-C27

8

N-'O (+I TR- P-P- -ADENINENADP+10

)I _ ATPP

INz GLUTAMINER®®.R-ADENINEDEAMIDO-NAD

9

R -®R-ADENINENAD+

FIG. 5. Aerobic tryptophan catabolic pathway to NAD (adapted from Moat [77]). Enzyme designations areas follows: 1, tryptophan 2,3-dioxygenase; 2, kynurenine formamidase; 3, kynurenine 3-monooxygenase; 4, 3-hydroxykynureninase; 5, 3-hydroxy-anthranilate 3,4-dioxygenase; 6, spontaneous reaction; 7, QAPRTase; 8,De-NAD pyrophosphorylase; 9, NAD synthetase; 10, NAD kinase. Abbreviations: PALP, pyridoxal 5'-phosphate; PRPP, phosphoribosyl pyrophosphate; R, ribose; (), phosphate.

thesis of NAD. The biosynthesis of NAD fromtryptophan in mammalian cells and lower eu-caryotes is now well documented as to the natureof the intermediates involved and the enzymesused to catalyze the individual reactions. Mostofthe references to this earlier work are includedin the reviews by Chaykin (16) and Dalgliesh(23) and will not be reviewed here.The pathway of tryptophan catabolism is de-

scribed as aerobic due to the strict oxygen re-quirements of the first enzyme, tryptophan ox-

ygenase, and the third enzyme, kynurenine 3-hydroxylase (95). Remarkably, tryptophan oxy-genase has been very difficult to demonstrate inextracts of several lower eucaryotes known tosynthesize NAD from tryptophan. For instance,this enzyme defied detection in Neurospora un-til 1973, when Casciano and Gaertner finallyused a fluorometric assay system which involveda kynurenine formamidase-kynureninase-cou-pled reaction (11). In microorganisms which donot synthesize NAD from tryptophan but do

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actively catabolize tryptophan, tryptophan oxy-genase is readily demonstrable as well as induc-ible by tryptophan or a tryptophan metabolite(82, 88). There are a number of similarities be-tween the pathway from tryptophan to NADand the aromatic pathway involved with thecatabolism of tryptophan (74). For example, thefirst two enzymatic reactions are identical. Fur-thermore, kynureninase, of the catabolic path-way, and hydroxykynureninase, of the NADpathway, have similar substrate specificities.The possibility that the tryptophan-NAD path-way evolved from the tryptophan catabolicpathway is discussed in Evolutionary Aspects.Evidence is accumulating which also implicatesthe aerobic NAD pathway in the biosynthesis ofactinomycin D by Streptomyces parvullus andStreptomyces antibioticus (46, 60). The follow-ing microorganisms have been shown to use thetryptophan-NAD pathway: X. pruni (10), Neu-rospora crassa (69), aerobically grown Saccha-romyces cerevisiae (1), and S. antibioticus (70).One very interesting aspect of de novo NAD

biosynthetic pathways is that they all lead tothe formation of a common intermediate, QA(16, 38, 43, 85, 101). Furthermore, the series ofsteps involved in the conversion of QA to NADare identical in all of the species which havebeen examined (Fig. 2). Preiss and Handler, in1958 (85, 86), studied the pathway from NA toNAD in human erythrocytes and yeast. Theyisolated the intermediates and identified the en-zymes involved by using a combination of ra-dioisotopic labeling and biochemical techniques.The reaction sequence they discovered is re-ferred to as the Preiss-Handler pathway andoccurs as follows:

NA -- NAMN -- deNAD -* NAD

Andreoli et al. (4) showed that in E. coli, QA,not NA, is the precursor of NA mononucleotide(NAMN) on the de novo pathway to NAD. QAis converted to NAMN by means of the phos-phoribosyl pyrophosphate-dependent enzymeQAPRTase. NA is now known to be part of thepyridine nucleotide cycle (PNC) which recyclesa variety of degradative products of NAD me-tabolism back to NAD (34). Biosynthetic routeswhich lead to the synthesis of NAD do so bysupplying QA to the PNC (Fig. 2).Dahmen et al. (22) have studied purified de-

samido-NAD (deNAD) pyrophosphorylase frombrewer's yeast and E. coli. Their studies showthat, in addition to catalyzing the NAMN-to-deNAD reaction, this enzyme can use nicotin-amide mononucleotide (NMN) as a substrate tosynthesize NAD in vitro. The yeast enzyme syn-thesized deNAD 2.2 times faster than it synthe-sized NAD. The enzyme from E. coli, however,

had a rate of deNAD synthesis 17 times fasterthan that of NAD. They concluded that, at leastin E. coli, the reactivity of deNAD pyrophos-phorylase with NMN in vivo is not physiologi-cally significant.The final step of NAD biosynthesis in either

the de novo pathway or the PNC is the amida-tion of deNAD to form NAD. The enzyme cat-alyzing this step is NAD synthetase. Preiss andHandler have shown that amidation of deNADin yeast involves L-glutamine as the amide donor(86), whereas E. coli preferentially uses free NH3in this reaction (100).

Genetics of Anaerobic NicotinamideAdenine Dinucleotide Biosynthesis

The preponderance of information regardingthe genetics of anaerobic NAD biosynthesiscomes from work with E. coli. Mutants blockedin the de novo biosynthetic pathway before thePNC may be isolated as NA-requiring auxo-trophs (Fig. 6). Three classes of NA-requiringmutants (relevant loci originally designated nic)were discovered in E. coli K-12 and S. typhi-murium LT-2. Originally, classification of theseloci was based upon mapping data. The geneswere later designated nadA, nadB, and nadC toreflect their roles in the biosynthetic pathway toNAD. The genetic map positions of these loci(Fig. 6) are 16, 55, and 2 min, respectively, in E.coli (7, 111, 112) and 17, 58, and 3 units, respec-tively, in S. typhimurium (28, 67, 90, 102, 103).Chandler and Gholson (12), in 1972, demon-

strated the excretion of QA and the absence ofQAPRTase in E. coli nadC mutants (78). BothnadA and nadB mutants failed to excrete QAbut did possess QAPRTase activity. Extractsfrom nadC mutants were capable of synthesizingQA from the precursors DHAP and aspartate,whereas extracts from nadA and nadB mutantslacked this capability (12, 14, 15). Extracts fromnadA and nadB mutants were able to comple-ment each other in vitro, resulting in the syn-thesis of QA (110). Therefore, the nadA andnadB genes encompass what is referred to asthe QA synthetase system, and nadC is thestructural gene for QAPRTase. Though less ex-tensive, the work by Foster and Moat (28) pro-vides evidence for similar gene designations inS. typhimurium.Kerr and Tritz (61), through liquid matrix

cross-feeding experiments with E. coli mutants,determined nadA to be the first enzyme of theQA synthetase system and nadB to be the sec-ond. Wicks et al. (115) have performed moredetailed experiments which suggest that thefunctional order of nadA and nadB is reversed.They found that toluene-treated nadB cells in-cubated with supernatant culture fluid from

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90 FOSTER AND MOAT

/ S. typhimuriummarkers

80o E. co/i 9° 1 20

markers ch

'4~~~~~~

> Xi o~~~~~~temnato

FIG. 6. Genetic linkage maps of E. coli and S. typhimurium, showing the genetic loci involved in NADbiosynthesis and PNC metabolism. Locations of the E. coli markers are shown on the inside of the circle, andthose of S. typhimurium are shown on the outside of the circle. The darkened area represents the suggestedinverted region.

nadA cells could synthesize ["C]QA from["4C]aspartate. The reverse situation, in whichtoluene-treated nadA cells were incubated withsupernatant from nadB cells, proved unsuitablefor QA synthesis. Thus, nadA cells producedsome diffusible compound or compounds whichnadB cells could use to synthesize QA. Conse-quently, the nadB gene product is the first en-zyme of the QA synthetase system and the nadAgene product is the second (Fig. 3).

Tritz and Chandler (110), while performing invitro complementation assays between extractsof various E. coli Nad- mutants, discovered agene involved in the regulation of NAD biosyn-thesis. The procedure involved mixing extractsfrom two different Nad- auxotrophs and observ-ing whether QA was synthesized or not. Whenextracts of nadA mutants were combined withextracts from nadB mutants (nadA and nadBdesignations were originally based on mappingdata), the nadB mutants fell into two separatecategories: (i) those that would complement thenadA extracts and (ii) those that would not. The

second group appeared to be regulatory mutantsin that they expressed neither the nadA nor thenadB gene product. The gene involved wastherefore designated nadR. This gene could notbe separated from nadB by transductional anal-ysis, and abortive transduction experimentswere never performed (G. J. Tritz, personal com-munication). Therefore, the status of nadR as aseparate cistron remains to be proven. Tritz(109) attempted to examine the nature of thenadR locus by performing merodiploid analysis.Merodiploids with the genotype nadR+/nadRlost their requirement for NA, thus indicatingpositive regulation by the nadR gene product.The model presented in Fig. 7 attempts to illus-trate positive control while maintaining thebasic concept that the addition of end productresults in the repression of enzyme synthesis.Although one might expect some form of nega-tive control (see Regulation of De Novo Biosyn-thesis of Nicotinamide Adenine Dinucleotide),this model is feasible. Griffith et al. (41) report,however, that the nadR strain used by Tritz was

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nodS nodR nodA

nad5 nadR nodA

Diffusibleproduct

+Corepressor(probably NAD)

Inactive regulotor proteinFIG. 7. Postulated mechanism for the positive regulation ofnadA and nadB by the nadR gene product. (A)

A diffusible product of the nadR gene would serve to promote or induce transcription of the unlinked nadABoperon. (B) However, in the presence of the corepressor, most probably NAD, this diffusible product would beincapable of "turning on" the expression ofnadAB. mRNA, Messenger ribonucleic acid.

actually a nadB mutant, not a nadR mutant.Therefore, results obtained by using a merodi-ploid with an actual genotype of nadR+/nadRrequire confirmation. Griffith et al. suggest thatthere may not be a separate nadR gene. Theregulatory function may be part of the nadBprotein. Abortive transduction experiments per-formed between numerous nadB mutants of S.typhimurium (some of which appear to benadR) suggest the presence of a single gene atthis locus (J. W. Foster, D. M. Kinney, and A.G. Moat; unpublished data).

Regulation of De Novo Biosynthesis ofNicotinamide Adenine Dinucleotide

NAD, albeit an essential cofactor, is requiredby the cell in extremely small quantities. Lund-quist and Olivera (71) have determined that theintracellular concentrations of NAD and NADPduring exponential growth of E. coli are 1.3 x10-3 and 3.9 x 1O-4 M, respectively. Thus, strictcontrol must be exerted by the cell to maintainthese levels.Regulation of de novo biosynthesis of QA was

first studied by Saxton et al. (93), in 1968. Theyreported that extracts prepared from E. coligrown in the presence of NA exhibited a de-creased capacity for QA synthesis, indicating arepression of the QA synthetase system by NAor a product formed from NA. In comparison,NA added to the growth medium had no effecton QAPRTase activity. Chandler and Gholson(12) further demonstrated that the quantity of

QA excreted by nadC mutants incubated in aspecial secretion medium was directly related tothe presence or absence of NA in the prepara-tory growth medium. The accumulated data def-initely indicate a repressive type of regulationover the QA synthetase system. The QA synthe-tase system may be subject to feedback inhibi-tion as well as end product repression. In vitroassays with crude (13) and partially purified (41)preparations indicate that concentrations of ox-idized NAD (NAD+) and reduced NAD(NADH) which approximate intracellular con-ditions (3.33 mM) strongly inhibit QA synthe-tase activity. Since equivalent levels of NADP+and NADPH did not have as dramatic an effect,these compounds do not seem to play a substan-tial role in the modulation of QA synthetaseactivity. In E. coli, QAPRTase does not appearto be under repression-derepression control orfeedback inhibition (35, 93). In contrast,QAPRTase synthesis is repressible in Bacillussubtilis (35, 93).

Tritz and Chandler (110), in 1973, reportedthe presence in E. coli of a gene involved in theregulation of NAD biosynthesis. Extracts fromcertain putative nadB mutants of E. coli failedto complement extracts of nadA mutants for invitro synthesis of QA. These particular nadBmutants were redesignated nadR by virtue ofthe fact that the lesion appeared to affect thesynthesis ofboth nadB and nadA gene products.Merodiploid analysis of these regulatory mu-tants by Tritz (109; see Genetics of AnaerobicNicotinamide Adenine Dinucleotide Biosyn-

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thesis) appeared to show positive regulation ofNAD biosynthesis by a diffusible product of thenadR gene. Griffith et al. (41), however, reportedthat the nadR mutant used in the study by Tritz(109) was actually a nadB mutant. Thus, thenature of the nadR locus and whether it isindeed distinct from nadB remain to be clarified.In their discussion, Griffith et al. (41) mentionthat the nadB protein binds to an NAD-agaroseaffinity column. This observation suggests thatthe nadB protein may possess regulatory fea-tures consistent with those of an aporepressor.An autoregulatory type of control system wherethe product of the nadB cistron serves to regu-late the expression of the noncontiguous nadABoperon seems reasonable. Thus, nadR mutantsmay represent a specific class of nadB muta-tions. However, QA synthetase also exhibitsfeedback inhibition (41, 93), and binding ofNADto the nadB protein may simply reflect this fact.Experiments designed to determine whether anadB protein isolated from an alleged nadRstrain can bind to the aforementioned NAD-agarose column could prove useful in assessingthe validity of the autoregulation theory.

X. pruni utilizes the tryptophan pathway forNAD biosynthesis, as mentioned above. Brownand Wagner (10) studied the regulation of thetryptophan pathway in this species. They ob-served coordinate induction of tryptophan pyr-rolase (tryptophan oxygenase), kynurenine for-mamidase, and kynureninase by L-tryptophan.L-Kynurenine was not effective as an inducer,which suggests that the effect is specifically dueto L-tryptophan. Subsequent work by these au-thors revealed that tryptophan pyrrolase is feed-back inhibited by NADH and NADPH as wellas by NAMN and anthranilic acid (114). It is ofinterest that the oxidized forms of the coen-zymes, NAD+ and NADP+, were ineffective asfeedback inhibitors of this enzyme. The otherenzymes of the tryptophan-NAD pathway werepresent, but no evaluation regarding their regu-lation was made (10). Remarkably, in this reportevidence is presented which suggests the pres-ence of a tryptophan-NAD pathway that utilizesanthranilic acid as an intermediate. NA-requir-ing mutants were isolated which could not growon tryptophan but could substitute anthranilatefor the NA requirement. The existence of analternate pathway involving anthranilic acidmay explain why this compound can inhibittryptophan pyrrolase activity. It should bestressed that no enzymological evidence existsfor this pathway as yet.

N. crassa also uses the tryptophan-NADpathway. Some studies have been conducted onthe regulation of this system. Lester (69), in

1971, suggested that tryptophan oxygenase maybe the rate-limiting step for NAD biosynthesisin N. crassa. He demonstrated repression andfeedback inhibition (9, 69) by nicotinamide(NAm) or, more likely, NAD. One questionwhich has persisted throughout the literature iswhether or not kynureninase, whose substratesinclude hydroxykynurenine, can substitute for ahydroxykynureninase in NAD biosynthesis.Gaertner et al. (32) have demonstrated a hy-droxykynureninase activity distinct from kynu-reninase. The hydroxykynureninase was shownto be constitutive in nature, whereas the kynu-reninase was inducible. Since a hydroxykynu-reninase would be involved with the synthesis ofan essential cofactor, NAD, the presence of aconstitutive enzyme would seem desirable. Onthe other hand, a constitutive kynureninase,which is involved only with the catabolism oftryptophan, would appear wasteful.

S. cerevisiae is an interesting species in thatit possesses both an anaerobic pathway and anaerobic pathway for NAD biosynthesis (1).Growth under aerobic conditions greatly favorsthe aerobic production ofNAD from tryptophanover the anaerobic route. One explanation forthis phenomenon has been offered by Schott etal. (94). They have demonstrated an apparentinduction of L-kynurenine 3-hydroxylase by ox-ygen, which is not observed in other systems,such as that of N. crassa (95). Growth of yeastunder anaerobic conditions yielded extractswhich possessed very little of this activity (0.026mU/mg of protein). A 10-fold increase in L-kyn-urenine 3-hydroxylase activity occurred whencells were grown aerobically. Work by Heilmannand Lingens (45) shows that 3-hydroxyanthran-ilate oxygenase of S. cerevisiae is not repressedor feedback inhibited by NAD or induced bytryptophan. Substrate concentrations higherthan 3 x 10-4 M, however, inhibited the activityof this enzyme. Thus, these two activities mayserve a major regulatory function in preventingan oversupply ofNAD under aerobic conditions.Ahmad and Moat (1) have shown that moreNAD is synthesized aerobically than anaerobi-cally. Thus, induction of L-kynurenine 3-hydrox-ylase by oxygen would lead to an increase inhydroxyanthranilate and NAD levels, assumingkynurenine 3-hydroxylase is a rate-limiting stepin this pathway. Too high a level of 3-hydrox-yanthranilate would inhibit the activity of 3-hydroxyanthranilate oxygenase, thus decreasingNAD biosynthesis.Regarding the question of kynureninase ver-

sus hydroxykynureninase activities and theirroles in NAD biosynthesis, Shetty and Gaertner(98) have discovered that S. cerevisiae only has

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a constitutive hydroxykynureninase. This spe-cies does not appear to degrade tryptophan viathe tryptophan-anthranilate cycle and thereforedoes not possess a kynureninase. This is in con-trast to N. crassa, which has both an induciblekynureninase and a constitutive hydroxykynu-reninase.

PYRIDINE NUCLEOTIDE CYCLEMETABOLISMBiochemistry

Recycling pathways, collectively known asPNCs, function in a majority of the speciesstudied. The basic outline for these cycles in-cludes the degradation of NAD to NAm, theconversion of NAm to NA, and finally the syn-thesis ofNAMN, an intermediate in the de novopathway to NAD, from NA. Cells that possessan intact PNC, and many cells that do not, usethe Preiss-Handler pathway to synthesize NADfromNA (52,85,86). Similarly, NAm deamidase,whose enzymatic function is to convert NAm toNA, is also present in all cells that can com-pletely recycle NAD (52, 55, 92, 99, 106) throughNA. The pathway of degradation from NAD toNAm, however, varies among different species.A majority of the eucaryotic species examinedcontain NAD glycohydrolase (2, 40, 57, 92, 108,120), which catalyzes the following reaction:

NAD+-- NAm

+ adenosine 5'-diphosphate-ribose

Figure 8 illustrates the five-membered PNC(PNC V) which uses this enzymatic activity tocleave NAD for recycling. NAD glycohydrolaseis relatively rare in procaryotic systems. An ex-tensive search was made for NAD glycohydro-lase in E. coli, but no such activity has beenfound (5). Species that lack NAD glycohydrolaseappear to recycle NAD by first degrading thecofactor to NMN and subsequently convertingthe NMN to NAm. There has been some con-troversy concerning which enzyme or enzymesare used by E. coli to generate NMN from NAD.Procaryotic deoxyribonucleic acid ligase (68, 80),which repairs single-stranded deoxyribonucleicacid nicks, uses NAD as a substrate in the re-action shown in Fig. 9. Manlapaz-Fernandez andOlivera suggest a major role for this enzyme inNAD turnover by E. coli (75). They report, asunpublished data, a reduced turnover of NADin a temperature-sensitive deoxyribonucleic acidligase mutant. However, E. coli, yeast (22, 50),and apparently S. typhimurium (26) also containNAD pyrophosphatase which can also degradeNAD to NMN and adenosine 5'-monophos-phate. These two enzyme activities probably

QA -NAMN--+deNAD - NAD

I/IPNCVY

N A,( NAmFIG. 8. PNC V.

1111111 11111111111 1 0O° , l I II l

LJ%H

NAm AdR R

T1 1 11 1 1 11 11 1 1

NAm AdI I

FIG. 9. Repair reaction catalyzed by deoxyribo-nucleic acid ligase. Abbreviations: R, ribose; (D),phosphate; Ad, adenine.

account for the majority of NAD hydrolysis inE. coli and presumably S. typhimurium. Anenzyme has been discovered in E. coli (5), Azo-tobacter vinelandii (49), and S. typhimurium (J.W. Foster and A. G. Moat, unpublished data)which specifically converts NMN to NAm. Thisenzyme, NMN glycohydrolase, occurs both in-tracellularly and membrane bound in E. coli (5).The enzyme from A. vinelandii has been puri-fied and studied quite extensively by Imai (49).His results show a pH optimum of between 8.5and 9.0 and a temperature optimum of 39°C.Data presented also reveal activation of thisNMN glycohydrolase by guanosine 5'-triphos-phate, deoxyguanosine 5'-triphosphate, and gua-nosine 5'-tetraphosphate, with KA values of0.025, 0.080, and 0.020 mM, respectively. Theother procaryotic NMN glycohydrolases havenot been examined as comprehensively, but theydo not appear to exhibit this activation by gua-nosine nucleotides.NAD pyrophosphatase and NMN glycohy-

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drolase, in essence, replace NAD glycohydrolasein the overall functioning of the PNC in mostprocaryotic species. Figure 10 illustrates the six-membered PNC (PNC VI), which includes theaforementioned enzymatic activities.Mutants of E. coli deficient in the de novo

biosynthesis of NAD are capable of growing onmedia supplemented with either NA or NAm.NA supports growth via the Preiss-Handlerpathway, and NAm supports growth throughconversion, by NAm deamidase, to NA (105).Sundaram et al. (106) proved this by isolating aNad- mutant that would grow on NA but failedto grow on NAm. Analysis of cell-free extractsrevealed that this mutant lacked NAm deami-dase activity.The first direct evidence for a functional PNC

in vivo came from Andreoli et al. (3) in 1969. Amutant of E. coli lacking NAm deamidase, afterincubation with ['4C]NA, accumulated ['4C]-NAm in the culture medium. They proposedthat the added NA was metabolized to NAD,which was subsequently degraded to NAm.Since the NAm could not be recycled, it wasexcreted into the culture medium. Control ex-periments, using a strain with an intact PNC,did not accumulate any ['4C]NAm. Manlapaz-Fernandez and Olivera (75) further examinedthe recycling of NAD by E. coli by means ofpulse-chase labeling experiments with differen-tially labeled NA and adenine. Cells grown inthe presence of ['4C]adenine and [3H]NA weretransferred to cold medium, and the "4C-3H ratioof intracellular NAD was measured at varioustime intervals. Wild-type E. coli displayed adecrease in the '4C-3H ratio, indicating the pref-erential loss of adenine label due to NAD turn-over. They also observed almost total conser-vation of the pyridine moiety in NAD duringthis recycling process. On the other hand, amutant lacking an intact PNC VI as a result ofNAm deamidase deficiency also exhibited a pref-erential loss of adenine label compared withniacin (NA) label from the pyridine nucleotidepool. The conclusion drawn form this experi-ment was that an alternate PNC, which doesnot utilize NAm as an intermediate, must existin E. coli. Evidence for a similar alternativePNC in S. typhimurium was first provided whena mutant blocked in the de novo pathway atnadA and in PNC VI at NAm deamidase(pncA) grew well on exogenously suppliedNMN. An alternate PNC had already been dis-covered in Clostridium sticklandii (31), A. vi-nelandii (48), and Propionibacterium sher-manii (Propionibacterium freudenreichiisubsp. shermanii) (29). The PNC in these spe-cies involves NMN deamidase, which specifi-cally deamidates NMN to form NAMN and

therefore bypasses NAm and NA (PNC IV [Fig.11]). A similar activity has recently been re-ported in S. typhimurium (62) and E. coli (75).The enzyme from S. typhimurium seems to dif-fer quite dramatically from that of P. shermanii(30) and A. vinelandii (48). The enzymes fromP. shermanii and A. vinelandii exhibit classicMichaelis-Menten kinetics and pH optima of 5.6and 7.0, respectively. The Salmonella NMNdeamidase seems to exhibit sigmoid kineticssuggestive of allosterism and has a pH optimumof 8.7. The purified enzyme from E. coli has apH optimum of 9.0 and appears to exhibit linearkinetics (B. M. Olivera, D. Hillyard, P. Manla-paz-Ramos, J. Imperial, and L. J. Cruz, personalcommunication). Substantial evidence is accu-mulating which shows that both PNC IV andPNC VI appear to function in vivo, with PNCIV being the predominant PNC. A strain of E.coli was found to recycle NAD 72% via PNC IVand 28% via PNC VI (75). Similarly, severalstrains of S. typhimurium tested revealed rela-tive contributions toward NAD turnover of 60to 69% for PNC IV and 31 to 40% for PNC VI(J. W. Foster and A. M. Baskowsky-Foster, sub-mitted for publication).Although the discovery ofNMN deamidase in

both S. typhimurium and E. coli proves thereare two PNCs, there is evidence in the literaturewhich appears to contradict this finding. Ghol-son et al. (37) have reported that a mutantblocked in the de novo pathway at nadB wascapable of growing in media supplemented withNAD, whereas a mutant blocked in both the denovo pathway at nadB and in the PNC at NAmdeamidase was incapable of growing in the samemedia. One would have expected the doublemutant to grow on NAD if the alternate, PNCIV, pathway existed in vivo. Recent studies withS. typhimurium may have resolved this paradox.

QA- NAMN ) deNAD- NAD

PNCVI j

NA4- NAm( -NMNFIG. 10. PNC VI.

QA-NAMN -*deNAD - NAD

PNC IV

NMNFIG. 11. PNC IV.

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Differentially labeled NAD transport experi-ments, using nadA and nadA pncA mutants,revealed that even though only nadA mutantstook up the pyridine moiety ofNAD, both nadAand nadA pncA cultures degraded NAD at thecell surface to NMN and adenosine 5'-mono-phosphate and accumulated these products ex-tracellularly (26). The data suggest that theNMN resulting from NAD degradation is readilytransported by NMN glycohydrolase and, in theprocess, converted to NAm. The fact that NMNglycohydrolase is associated with the membraneof E. coli (5) tends to support this theory. ForNMN to traverse the membrane intact, it mustapparently utilize a transport system other thanNMN glycohydrolase. Effective utilization ofthis system seems to occur when exogenousNMN levels reach 10' M or more. Thus, onecould predict that a large number of cells shouldbe capable of degrading extracellular NAD toyield a concentration ofNMN sufficient to per-mit entry by this alternate uptake system. Onceintracellular, the NMN could be recycled viaPNC IV andNMN deamidase. The result shouldbe growth of the double mutant on NAD. Thisexperiment was successfully performed with S.typhimurium (26). Subsequently, mutants un-able to transport NMN via this alternate systemhave been isolated and designated pyridine nu-cleotide uptake mutants (pnuA). This gene hasbeen mapped at 0 units via cotransduction withthe thr gene (26, 62). A model for the utilizationof exogenous NAD by S. typhimurium and E.coli is presented in Fig. 12.NADP is derived from NAD by an adenosine

5'-triphosphate (ATP)-dependent phosphoryla-tion catalyzed by NAD kinase. The reactionproceeds as follows:

NAD+ + ATP -- NADP++ adenosine 5'-diphosphate

Studies with S. cerevisiae suggest there arethree NAD kinases in this species. One is acytoplasmic enzyme specific for NAD and in-hibited by NADH; another is an NAD-specificmitochondrial enzyme; and the third, also mi-tochondrial, is specific for NADH (NADH ki-nase) (6). They appear to be separate and dis-tinct enzymes, as evidenced by (i) three differentrates of inactivation by heat and 3-(bromoace-tyl)-pyridine, (ii) different Km values, (iii) differ-ent pH optima of activity, (iv) sigmoid Michaelisplots found with the mitochondrial kinases, and(v) inhibition of NAD kinase by high substrateconcentrations. The control of NADP(H) syn-thesis is evidently very complex in this species.Part of the complex control could be in the formof allosteric modifiers, as suggested by the sig-moid kinetics of the mitochondrial kinases.Evidence indicating the presence of NAD ki-

nase in E. coli has been presented by Imsandeand Pardee (53) as well as Lundquist and Olivera(71). Although detailed enzymological studieswere not performed, this enzyme seems to beinhibited by high ATP levels (53). Lundquistand Olivera (71) have presented data which sug-gest that the interconversion of NAD andNADP is important in the regulation of PNCmetabolism. This aspect will be analyzed below(Regulation).

Pyridine Nucleotide Cycle GeneticsSundaram (105), in 1967, isolated a mutant of

E. coli which lacked NAm deamidase. This en-zyme converts NAm to NA and functions as an

N MN NAm INSIDE

NAD PNC NA

dNAD NAMN

FIG. 12. Model for utilization of exogenous NAD by S. typhimurium and E. coli. Abbreviations: AMP,adenosine 5'-monophosphate; dNAD, deNAD.

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integral part of PNC VI. Penicillin selectionprocedures were used to isolate nadC mutantscapable of growing on NA but incapable of grow-ing on NAm. The mutation is located at 39 minon the E. coli linkage map (7, 24) and is desig-nated pncA. Extracts from pncA mutants, aspredicted, lackedNAm deamidase. SimilarpncAmutants were isolated in S. typhimurium on thebasis of their resistance to the NAm analog 6-amino-NAm. In contrast to E. coli, the pncAlocus mapped at 27 units in this species (Fig. 4),between the gal and trp operons (26). This locusin E. coli resides between the trp and his op-erons. Thus, pncA appears to reside within thelarge inverted region described in the reviews bySanderson and Hartman (90) and Riley andAnilionis (87). However, placement of pncA inS. typhimurium at 27 units suggests that theinversion extends beyond the 10 units proposedto nearly 14 units (Fig. 6). Further experimen-tation with mutants in this region is needed toconfirm this theory.Pardee et al. (83), interested in how microcon-

stitutive enzymes are maintained at low levels,isolated a mutant of E. coli capable of growingon NAm as the sole source of nitrogen. Themutant was a hyperproducer of NAm deami-dase, and the relevant gene was designatedpncH. This gene mapped at 39 min in E. coliand was cotransducible withpncA. The purposebehind the search for this type of mutant was tostudy the regulation ofNAm deamidase produc-tion. Even though this enzyme is constitutive innature, Pardee has postulated that enzymes pro-duced in low quantities may have developed asystem of regulation distinct from the classicinduction-repression control mechanisms whichutilize a diffusible regulatory element. Induc-tion-repression regulation appears too costly forthe cell when one considers the amount of en-zyme produced. For example, wild-type E. coliproduces NAm deamidase with a specific activ-ity of 3 nmol of NAm hydrolyzed per min permg of protein (24). This is a value several ordersof magnitude below those of many enzymes in-volved in other major bacterial pathways. ThepncH mutants produced enzyme with a specificactivity up to 50 times normal levels. This wasshown to be a true increase in the amount ofenzyme produced and not simply a more activeenzyme. Although the actual cause for this in-creased synthesis is not known, one can specu-late that perhaps a promoter "up" mutationoccurred which would allow for more frequentmessenger ribonucleic acid initiation by ribonu-cleic acid polymerase.

R. Lemmon, J. Rowe, and G. Tritz reported(Abstr. Annu. Meet. Am. Soc. Microbiol. 1977,K229, p. 224) that mutants resistant to the NA

analog 6-amino-NA lack NAPRTase activity.The relevant gene in these mutants was desig-nated pncB. Attempts to map these mutants byconjugation have been frustrated by the highfrequency of spontaneous resistance associatedwith this analog. First reports, however, placedpncB between 20 and 30 min on the E. colilinkage map.

Foster et al. have isolated pncB mutants of S.typhimurium which also lack, or possess reducedlevels of, NAPRTase (26). However, throughcotransduction and mutagenesis they success-fully introduced nadA and nadB mutations intothese pncB mutants. This served to eliminatethe problem of the development of spontaneous6-amino-NA-resistant mutants during mappingprocedures. ThepncB locus has been mapped inthis species via conjugation and was found toreside near 25 units (Fig. 6).

RegulationNAD, as a cofactor, is used for catabolic en-

ergy-yielding oxidations, whereas NADP servesas the source of biosynthetic reducing power.Since NAD also serves as a substrate in cellularmetabolism, it is reasonable to expect that thesynthesis and breakdown of NAD and NADPare carefully regulated. Imsande, in the early1960s, first demonstrated the regulation of oneof the PNC enzymes in E. coli (50, 51, 53). Theactivity of NAPRTase in extracts prepared fromcells grown in medium containing NA was 100-fold lower than the activity found in extractsprepared from cells grown in NA-free medium.In contrast, this enzyme was not regulated byeither repression or feedback inhibition in Ser-ratia marcescens, B. subtilis, Torula cremoris,or Tetrahymenapyriformis (51). Presumably, inE. coli NA is converted via the Preiss-Handlerpathway to NAD. NAD would then function insome capacity to repress transcription of theNAPRTase structural gene, pncB. NAD cannotbe used in this type of study because exogenousNAD is not utilized directly by the cell, but mustbe degraded and recycled through the PNC (26,37).Imsande (51) in 1964 and Foster et al. (26) in

1979 have measured NAPRTase levels in S.typhimurium. Repression of this enzyme doesoccur, but at a lower level than with E. coli. Adetailed examination of NA concentration ver-sus NAPRTase activity revealed that repressiondid not occur until the NA concentration was atleast 7 x 10-7 M (27). Maximal repression oc-curred around 5 x 10' M NA. This study alsorevealed that in order to obtain repression withother intermediates of the PNC, they must beused at a concentration which will result in anadA mutant generation time of 65 min or less.

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Although the exact mechanism of repressionhas not been proven, there is evidence whichimplicates NAD as the true corepressor mole-cule. Experiments with labeled precursors invivo result in most, if not all, of the label residingin NAD and NADP (26, 72; Foster and Bas-kowsky-Foster, submitted for publication). Thisfinding suggests that intracellular levels of theother intermediates are too low to cause repres-sion. Furthermore, studies with S. typhimuriumhave shown that although NAm and NMN willcause repression of NAPRTase in a wild-typestrain, the introduction of apncA mutation pre-vents this repression (26). These NAm deami-dase mutants do not recycle exogenous NAm orNMN via PNC VI to NAD, suggesting thatrepression of NAPRTase by various PNC inter-mediates is the result of their conversion toNAD. The concentration ofNMN used was notsufficient to produce a nadA pnecA mutant gen-eration time of 65 min, which explains why therewas no repression with this nucleotide eventhough it could be recycled by PNC IV.The possibility of feedback inhibition byNAD

was explored by Imsande and Pardee in E. coli(53) and Foster et al. in S. typhimurium (26), butwith negative results. In light of the resultsobtained with tryptophan oxygenase, whereNADH but not NAD+ successfully inhibitedactivity (114), perhaps NADH should be exam-ined for any effect it may have on NAPRTaseactivity. Other PNC enzymes of E. coli, whenstudied, failed to show any regulation. Pardee etal. (83) and Baecker et al. (8) found no changein NAm deamidase activity when E. coli wasgrown in media supplemented with NA, NAm,NAD, or NADP. deNAD pyrophosphorylase,NAD synthetase, and NAD kinase activitiesfailed to show any decrease in activity whenthese enzymes were extracted from cells grownin 1o-4 M NA-supplemented minimal medium(53). These authors, therefore, suggested thatNAPRTase is one of the key control elements ofthe PNC.

Further evidence indicating the precise con-trol exerted over NAD metabolism comes fromLundquist and Olivera (71). Exponentiallygrown E. coli maintained a steady-state balancebetween the levels of NAD and NADP. In ad-dition, the breakdown of NADP to NAD wasfound to be one of the major processes whichdetermines the relative levels of NAD andNADP. Lundquist and Olivera (71) present amodel, based on labeling data, which representsthis steady state as follows:

NA- NAD NADPcell wall R2

R. is the rate of fonnation ofNAD from externalNA in each cell, R1 is the average rate of conve-sion of NAD to NADP per cell, and R2 is theaverage rate of breakdown of NADP to NADper cell. Interestingly, other intermediates of thePNC are present in such infinitesimal amountsthat the only pyridine compounds labeled aftera ['4C]NA pulse-chase experiment are NAD andNADP. Regulation, therefore, appears to in-volve the interconversion of NAD and NADP(the normal ratio of NADP to NAD being cal-culated at 0.3) and the regulation of NAD syn-thesis through the repression of QA synthetaseand NAPRTase. An imbalance in the ratio ofNAD to NADP due to excess NAD would resultin the repression of NAPRTase. Normal recy-cling of the NAD would occur due to the appar-ent constitutivity of the other PNC enzymes.The lowered NAPRTase activity would causethe loss of some NA through excretion andtherefore reduce the level of intracellular NAD.These events should restore the NADP-NADratio back to 0.3. Support for this theory isprovided by Wimpenny and Firth (118). Theyhave measured NAD(H) levels in E. coli andKlebsiella aerogenes (K. pneumoniae) duringtransition from aerobic to anaerobic growth.Rapid drops in NAD levels were observed whichcould not be explained by simple reduction toNADH. They presume that this loss is due toNAD turnover. This hypothesis is feasible, sinceboth cycles have been shown to function in vivoin E. coli (75) and S. typhimurium (Foster andBaskowsky-Foster, submitted for publication).However, Olivera has characterized a PolI-EndoI- strain of E. coli that does not appear torecycle intracellular NAD via PNC VI. (Oliveraet al., personal communication). Thus, a defini-tive role for PNC VI in regulating intracellularNAD levels remains to be established.The importance the PNC commands in the

regulation of NAD biosynthesis in E. coli isreported by McLaren et al. (76). They concludethat the de novo biosynthetic pathway is theleast preferred route for NAD biosynthesis,whereas the Preiss-Handler pathway takes prec-edence. At an NA concentration of 8 x 10-7 M,the average E. coli cell took up 90 molecules ofNA per s for NAD biosynthesis and synthesized340 NAD molecules per s de novo. Increasingthe NA concentration to 2 x 106 M resulted in215 molecules of NAD being derived from themedium per s and 215 molecules per s synthe-sized de novo. Finally, at 4 x 10-6M NA, endog-enous synthesis of NAD is completely sup-pressed. All of the intracellular NAD is thenderived from the medium. Their conclusion thatthe PNC takes precedence over de novo synthe-

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sis is supported by Chandler and Gholson (12,13), who demonstrated a repression of QA syn-thetase activity by NA (presumably throughconversion to NAD; see Regulation of De NovoBiosynthesis of Nicotinamide Adenine Dinucle-otide). Foster et al. (27) arrived at a concentra-tion similar to that mentioned above (5 x 106M NA) for the maximal repression of NA-PRTase in S. typhimurium.Some enzymes of the PNC have been shown

to be associated with the cell surface. NMNglycohydrolase (5) was shown to be an integralcomponent of the cytoplasmic membrane, andNAPRTase appears to be located within theperiplasmic space of E. coli (8). The study withNAPRTase utilized an osmotic shock treatmenttermed the ethylenediaminetetraacetate-lyso-zyme-freeze-thaw method to release NAPRTasefrom the periplasmic space. This procedure re-leased more enzyme as compared with sonicoscillation or alumina grinding. In addition,growth of E. coli supplemented with NA pro-duced only a fourfold reduction in total enzymeactivity, compared with the 100-fold decreasereported by others using alumina grinding tech-niques (51, 53, 93). Thus, the repression of NA-PRTase in E. coli is not as strong as originallybelieved.NAD pyrophosphatase or an enzyme similar

in function may also be associated with the cellmembrane, at least in S. typhimurium (26). S.typhimurium fed ['4C]NAD rapidly degradedthis NAD to [14C]NMN and adenosine 5'-mono-phosphate, both of which accumulate extracel-lularly. This degradation and extracellular ac-cumulation occurred even in pncA mutantswhich did not readily incorporate '4C intracel-lularly from exogenously supplied ["4C]NAD.These findigs suggest that NAD degradationoccurs at the membrane level. Thus, in sum-mary, there is evidence suggesting a membraneassociation for two or possibly three PNC en-zymes in E. coli and S. typhimurium.

Organisms of Special InterestHaemophilus. It is well known that Haemo-

philus influenzae and H. parainfluenzae re-quire NAD for growth (25, 73). Neither NA norNAm can substitute for NAD. H. haemoglo-binophilus, however, does not require NAD asa growth factor (89). Kasarov and Moat (58)have demonstrated that this species synthesizesNAD from NAm by the following series of re-actions:

(i) NAm + 5-phosphoribosyl pyrophosphate

+ ATP NMNNAmPRTase

(ii) NMN + ATP NADdeNAD (NAD) pyro-

phosphorylase or NMNadenylyltranferase

This organism could not convert either QA orNA to NAD, indicating the lack of either a denovo biosynthetic pathway or a Preiss-Handlerpathway.Mycobacteriunm A test commonly used to

distinguish human strains of M. tuberculosisfrom bovine strains and the atypical mycobac-teria is the NA test (63, 64). Human varieties ofM. tuberculosis accumulate large quantities ofNA in the culture medium, whereas other my-cobacteria do not. A study by Kasarov and Moat(57) revealed that extracts prepared from a hu-man strain of M. tuberculosis had very highlevels of NAD glycohydrolase and NAm deami-dase when compared with the bovine strain.Furthermore, whereas enzyme preparations ex-hibited activity with regard to the enzymes ofthe biosynthetic pathway from QA throughNAD, NAPRTase levels were extremely low orabsent. The human strain of M. tuberculosisrapidly degrades NAD to NA but cannot recyclethe NA to NAD. The NA then accumulatesextracellularly. The bovine strain, also with lowNAPRTase activity, does not degrade NAD asrapidly, thereby accumulating much less NA.

Procedures used to assay NAD glycohydro-lase from M. tuberculosis include a heat activa-tion step (108). Gopinathan et al. (39, 40) haveshown that heating the extract destroys an in-hibitor of mycobacterial NAD glycohydrolase.In fact, one of the more plausible theories ofisoniazid inhibition involves the binding of theNAD glycohydrolase inhibitor by isoniazid, thusreleasing the enzyme activity. The increasedactivity would consequently deplete the intra-cellular NAD pool (101). The presence of NADglycohydrolase and the absence of a PNC appearto provide a logical explanation for the fact thatisoniazid is effective against M. tuberculosis andnot against a variety of other bacteria.Clostridium butylicum. C. butylicum (C.

acetobutylicum), a strict anaerobe, synthesizesNAD from aspartate and formic acid as men-tioned previously. C. butylicum also accumulatesNA in culture media (54). In contrast to M.tuberculosis, however, this NA is not derivedfrom the degradation of NAD. Rather, it is theresult of NAMN and deNAD degradation (59).The pathway of degradation was shown to be asfollows:

deNAD -- NAMN -- NA-riboside -- NA

Unexpectedly, 5 to 10 mM ATP stimulateddegradation, whereas 20 mM ATP was required

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to reduce this degradation and allow conversionof NAMN and deNAD to NAD. Attempts todemonstrate recycling of NA were unsuccessful,indicating the lack of NAPRTase in this species(13,59). NAD pyrophosphatase and NAm deam-idase activities were found (59), but no means ofconverting NMN to NAm was observed. Al-though it appears unusual for a species to irre-versibly degrade a cofactor as important as

NAD, this degradation may represent a primi-tive mechanism for metabolic control over theintracellular concentration of this cofactor (seePyridine Nucleotide Cycle Metabolism, Regu-lation). Other studies have shown that C. stick-landii extracts have an NMN deamidase activ-ity which converts NMN to NAMN and thusrecycles NAD (31). C. butylicum was not exam-

ined for this enzyme. Other clostridia and anaer-

obic genera should be examined for this type ofrecycling capability.Azotobacter vinelandii Reports from Imai

(48, 49) reveal that A. vinelandii, an aerobic,nitrogen-fixing species, possesses a PNC IV anda PNC VI similar to those of E. coli and S.typhimurium. The following activities have beenmeasured: QAPRTase, NAPRTase, NAMN ad-enylyltransferase, NAD synthetase, NAD ki-nase, NAD glycohydrolase, NAD pyrophospho-rylase, NMN deamidase (48), and NMN glyco-hydrolase (49). One can see from this list ofenzymes that in addition to PNC IV and PNCVI, there is a PNC V which utilizes NAD gly-cohydrolase. This is the only species in whichenzymes for all three types of PNC have beendemonstrated.Lactobacillus and Leuconostoc. Members

of Lactobacillus and Leuconostoc appear inca-pable of synthesizing NAD de novo. However,different species have developed different path-ways to satisfy their pyridine nucleotide require-ment (79). For example, Leuconostoc mesenter-oides specifically requires NA, but Lactobacil-lus fructosus (Lactobacillus fructivorans) willonly use NAm to synthesize NAD. Lactobacil-lus plantarum and Lactobacillus casei, how-

ever, can use either NA or NAm. A study byOhtsu et al. (79) in 1967 explains this phenome-non on the basis of each species' complement ofPNC enzymes. Table 3 summarizes their data.One can see that those species which specificallyrquire NAm convert NAm to NAD via NMN ina manner similar to that shown for H. haemo-globinophilus (58). Lactobacilli which only useNA lack NMN pyrophosphorylase and NAmdeamidase. Species which use either NA orNAm appear to have an intact PNC. The path-way from NAm to NAD via NMN may beexpected to operate in all species which are

strictly dependent upon NAm for growth. How-ever, other species of this type, e.g., Pasteurellamultocida (65) and Haemophilus species whichdo not require NAD for growth (121), have notbeen studied with regard to the mechanism ofconversion of NAm to NAD.

EVOLUTIONARY ASPECTSEvolution of Biosynthetic Pathways

Since NAD is an essential cofactor in bothaerobic and anaerobic processes, a pathway forits synthesis must have existed in primitive cel-lular organisms. Furthermore, since the earth'senvironment was initially anaerobic, it followsthat the first biosynthetic pathway to NADcould not have involved molecular oxygen. Thus,a de novo biosynthetic pathway similar to theEscherichia-Salmonella aspartate pathway orthe clostridial aspartate pathway would fulfillthe requirements of a primordial system.

Gaertner and Shetty (33) have discussed thepossible evolution of the aerobic tryptophanpathway to NAD. They suggest five steps in theevolutionary process. Stage I is the developmentof the anaerobic biosynthetic pathway describedabove. Stage II is the appearance of the induc-ible kynureninases necessary for the catabolismof tryptophan as a carbon source. The subse-quent formation of the aerobic NAD biosyn-thetic pathway from the inducible tryptophancatabolic enzymes constitutes stage III. Stage IVincludes the further refinement of the aerobic

TABLE 3. PNC enzymes present in various microorganisms displaying different specificities for NA or NAm

Enzyme present

Species Requirement NAD

NAPRTase NAm NMN pyro- (deNAD)deamidase phosphorylase pyrophos-

phorylase

L. mesenteroides NA + - - +L. fructosus NAm - - + +L. plantarum NA or NAm + + - +L. casei NA or NAm + + +/-a +

a +/-, L. casei possessed barely measurable levels ofNMN pyrophosphorylase activity. (Adapted from Ohtsuet al. [791.)

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100 FOSTER AND MOAT

pathway, elimination of the inducible kynureni-nases, and loss of the anaerobic NAD biosyn-thetic pathway; interestingly, S. cerevisiae andpossibly other fungi appear to be transitionalspecies, possessing both anaerobic and aerobicpathways to NAD (1, 98) but lacking an induc-ible kynureninase (98). Gaertner and Shetty sug-gest that the final stage, stage V, is the completeelimination of all de novo NAD synthesizingcapacity, e.g., H. influenzae.

Evolution of the Pyridine Nucleotide CycleEvery species that has been studied regarding

NAD turnover, be it procaryote or eucaryote,has the ability to degrade NAD. Not all appearto be fully capable of recycling the degradationproducts, but degradation products are formed.One could conclude that the turnover of NAD isvery important to the cell. This importance ismost probably manifested in the maintenance ofa certain NADP-NAD ratio, as suggested byLundquist and Olivera (71), as well as the main-tenance of a specific quantitative level of NAD,as suggested by Foster et al. (27).

Several enzymes involved with PNC metabo-lism appear to be the result of gene duplicationand divergent evolution (87). For example, NA-PRTase and QAPRTase are very similar withregard to their substrate specificities, the onlydifference between NA and QA being an extracarboxyl group on QA. NAm deamidase andNMN deamidase perform similar functions, asdo NAD pyrophosphatase and deNAD pyro-phosphorylase. In addition, NAD glycohydro-lase could have arisen from a modification ofNMN glycohydrolase or vice versa. Speculationas to the order in which these evolutionarychanges took place would be unwarranted at thispoint. Suffice it to say the changes did occur andhave been maintained. Maintenance of a geneticduplication or alteration, however, implies a cer-tain amount of selective advantage associatedwith it. Why, though, should such organisms asPropionibacterium (30), E. coli (75), and S. ty-phimurium (62) possess two recycling pathways(PNCs IV and VI) and A. vinelandii possessthree (48,49)? The major difference between thethree cycles is that PNC IV preserves the ribose-5-phosphate moiety of NMN, whereas PNC Vand PNC VI promote the turnover of this com-pound with subsequent ATP-dependent replace-ment. The suggestion has been offered that PNCIV could be advantageous due to its minimalexpenditure of energy (62). PNC V and PNC VI,on the other hand, appear to be most suited forthe transportation and conversion of preformedpyridine compounds found in the environment.Results from Manlapaz-Fernandez and Olivera(75), B. M. Oliver, D. Hillyard, P. Manlapaz-

Ramos, J. S. Imperial, and J. Cruz (manuscriptin preparation), and Foster and Baskowsky-Fos-ter (submitted for publication) support the con-cept of an intracellular preference for recyclingNAD via PNC IV. PNC VI, although predomi-nantly used for the transport of preformed pyr-idine compounds, may also be used to fine-tuneintracellular NAD levels through the repressionofNAPRTase. One question presents itself. Whydoes a genus, such as Azotobacter, possessthree PNCs? Much more research will be re-quired before a definitive conclusion can bemade. However, one may suggest that Azoto-bacter represents an evolutionary branch pointin PNC metabolism.

Nevertheless, all of the enzymes involved inthe recycling of NAD as well as the regulationassociated with them represent a phenomenaleffort by the cell to preserve a compound, NA,whose primary purpose involves the biosyn-thesis of NAD (Friedmann and his co-workers[29a, 30] have shown that in some microorga-nisms NAMN is also involved in the synthesisof cobalamin). Perhaps it is just coincidence thatthese enzymes and controls are present. Thenagain, perhaps it is not. There may be essentialfunctions of NAD or its recycling yet to bediscovered in both procaryotic and eucaryoticmicrobial metabolisms.

SUMMARYFrom the various studies that have been re-

viewed with regard to the biosynthesis of NAD,it can be generally stated that mammalian cellsof most types, yeasts and molds metabolizingunder aerobic conditions, and one or two unu-sual genera of bacteria are all capable of con-verting the ring carbon and nitrogen of trypto-phan to the pyridine ring ofNAD (Fig. 13). Mostof the procaryotic species that have been exam-ined to date utilize aspartate and DHAP to fonnthe pyridine ring. The anaerobe C. butylicumappears to be the only species that utilizes astepwise pathway involving the condensation offormate with aspartate to form N-formylaspar-tate and further condensation with acetyl coen-zyme A to yield the pyridine ring of QA. QA isthe common intermediate in all of these path-ways, and the steps from QA to NAD appear tobe identical in all species capable of convertingthis compound to NAD.Among species that require either NA or

NAm, those that are not specific in their require-ment (i.e., that can use either NA or NAm tosatisfy their NAD requirement) convert NA toNAMN via NAPRTase. NAm is deamidated toNA. Species that exhibit a specific requirementfor NAm convert this compound to NMN andthen to NAD. Although the NMN pathway ofNAD synthesis appears to be relatively rare in

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NAD BIOSYNTHESIS AND PNC METABOLISM 101

BACTERIAMycobacterium tuberculosiJ(scherichia co/iSu/mone//l typhimuriumBocillu# sublilis

FUNGI (ANAEROBICALLY)SO*E PLANTS

H2 COH COOHO-C H 'H

I +H2C-O-® SH

H2N COOHDHAP ASPARTATE

COOHHCH

HCOOH + I

H2N OOH

FORMATE ASPARTATE

cII H2CHCOOH

TRYPTOPHANMAMMALS

6 * YEASTS,MOLDSSTEPS# SOME PLANTS

* Xatehomoea prua/*

COOH

NiCOOHQUINOLINATE

CH3COSCoA

fOOHHCH

-~~~~~~~ ~~

HCO CH\N 'OOHH

N-FORMYLASPARTATE

"COMMON PATHWAY(PREISS-HANDLER)

NAMN -'bDEAMIDO-NAD----*NAD4

NA

NAm

NMN

NAm

NA or NAm-requiring

organisms

ANAEROBIC BACTERIA (Clostridium buty/icum)

NAm-specificorganisms

Heemophi/ushaemog/obhinophilus

L octoboci//usfruclosus

FIG. 13. Summary ofNAD biosynthetic pathways. [XI represents unknown intermediate(s). Abbreviations:,)phosphate; CoA, coenzyme A.

microbial cells, it is present in a wide variety ofmammalian cells and is intranuclear in location.

Species capable of degrading NAD and recy-cling the various degradation products to reformNAD utilize one or more alternative pathwaysreferred to collectively as PNCs. Most procar-yotes examined are capable of degrading NADto NMN and then to NAm and NA. Final recy-cling of NA to NAD occurs via the Preiss-Han-dler pathway. In E. coli, S. typhimurium, Azo-tobacter, and Propionibacterium, an alternatecycle (PNC IV) is present as a result of theactivity of NMN deamidase. M. tuberculosisappears to be one of the few procaryotic speciesthat degrades NAD via NAD glycohydrolase.However, this species lacks NAPRTase activityand, instead of recycling NA, excretes NA intothe medium. Most eucaryotes, including yeastsand molds, degrade NAD via NAD glycohydro-lase. The NAm produced is deamidated to NAand recycled to NAD via the Preiss-Handlerpathway.Although the various pathways leading to the

formation of NAD appear to be rather complex,this is not altogether surprising in view of theessential nature of NAD in cellular metabolism.What is surprising is the relatively late devel-opment of interest in detailed studies of thesystems and regulation involved in the biosyn-thesis and metabolism of NAD. A much broadersurvey of the numerous microbial genera would

be of interest to determine the degree of conser-vation or divergence which may have occurredin the evolutionary development of pathways ofsynthesis and metabolism of NAD.

Several aspects of NAD metabolism have yetto be resolved. The problem which has attractedthe most attention, and yet has proven to be themost frustrating, concerns the isolation andidentification of the intermediate(s) involved inthe aspartate-DHAP pathway in procaryotes.Although several possible structures have beenproposed, none has been well characterizedchemically. Progress has been achieved in thisarea, however, in that several potential com-pounds have been isolated, and some defmiitiveanswers should be forthcoming.Another question which requires a satisfac-

tory explanation is the necessity of multiplepathways for the recycling of NAD, particularlywithin a single species. Is NAD so precious acofactor that evolutionary selective processesresulted in the emergence of several recyclingcapabilities? The most obvious rationalizationwould be that the ability to preserve the pyridinering is essential to optimal metabolic capability,particularly if there are surges in the require-ment for NAD as a substate. However, a role forthese cycles in the regulation of intracellularlevels of NAD cannot be overlooked. Furtherinvestigation with mutants blocked in each ofthe PNC pathways should provide a fruitful

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avenue of approach to this problem.Further investigation will be necessary to

characterize the genetic loci which code for theenzymes involved in NAD metabolism. Signifi-cant progress has been made in this regard withS. typhimurium and E. coli, but a great deal ofadditional work will be required to fully eluci-date the genetic control of pyridine nucleotidemetabolism. Additional studies with a completeset of mutants blocked in each of the steps inthe metabolic pathways would provide a morecomplete and accurate picture of the geneticsand regulation of NAD biosynthesis and metab-olism.

ACKNOWLEDGMENTSWe are indebted to H. Friedmann for his critical

reading of the manuscript and to R. K. Gholson andB. M. Olivera for their helpful discussion.

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