THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc Vol. 264, No. 6, Issue of February 25, pp. 3200-3205 1989

Printed in Li.S.A.

Structure and Properties of Malic Enzyme from Bacillus stearothermophilus”

The malic enzyme (EC 1.1.1.38) gene of Bacillus stearothermophilus was cloned in Escherichia coli, and the enzyme was purified to homogeneity from the E. coli clone. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, the enzyme cat- alyzes the decarboxylation of oxalacetate. The enzyme is a tetramer of M, 200,000 consisting of four identical subunits of M, 50,000. The pH optima for malate oxi- dation and pyruvate reduction are 8.0 and 6.0, respec- tively; and the optimum temperature is 55 O C . The enzyme strictly requires divalent metal cations for its activity, and the activity is enhanced 5-7 times by NHa+ and K+. Kinetic study shows that the values of the dissociation constant of the enzyme-coenzyme com- plex are 77 MM for NAD and 1.0 mM for NADP, indi- cating that the enzyme has a higher affinity for NAD than for NADP. The nucleotide sequence of the gene and its flanking regions was also found. A single open reading frame of 1434 base pairs encoding 478 amino acids was concluded to be that for the malic enzyme gene because the amino acid composition of the enzyme and the sequence of 16 amino acids from the amino terminus of the enzyme agreed well with those deduced from this open reading frame.

The malic enzyme ((S)-ma1ate:NAD’ oxidoreductase (ox- alacetate-decarboxylating)) catalyzes the NAD(P)-dependent oxidative decarboxylation of L-malate and has been classified into three groups (EC 1.1.1.38, 1.1.1.39, and 1.1.1.40) on the basis of its coenzyme specificity and its ability to catalyze the decarboxylation of oxalacetate. As the equilibrium of the reaction lies in favor of NAD(P)H formation, this enzyme is useful for colorimetric measurements of malate and also for NAD(P)H regeneration units in enzyme reactors (1).

The enzyme is widely distributed in various organisms such as bacteria (2-6), plants (7, 81, and higher animals (9-11). Thermostable malic enzymes have also been isolated from Sulfolobus solfataricus (3) and Clostridium thermocellum (4), but the enzyme from Bacillus stearothermophilus has not been reported. To understand the structure-function relationships of the malic enzyme and to use this enzyme as an NAD(P)H regeneration unit, we planned to clone the gene from B.

* This work was supported in part by Grant-in-Aid 63850191 from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) MI 9485.

$ To whom correspondence should be addressed Dept. of Fermen- tation Technology, Faculty of Engineering, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565, Japan.

stearothermophilus. The enzymes from thermophilic orga- nisms are generally stable and are easily purified from Esch- erichia coli cells having the genes of the enzymes. So far, little is known about the structure of malic enzymes of mesophiles and thermophiles. The only examples reported previously are the structures of murine and rat malic enzymes (9, 10). Cloning of the malic enzyme gene from B. stearothermophilus would enable us to find the primary structure of the enzyme and to obtain enough of the enzyme for study. Cloned genes can be used for investigation of the structure-function rela- tionship by protein engineering.

In this study, we first constructed a gene library of the B. stearothermophilus chromosome in E. coli and screened for the gene of the thermostable malic enzyme by qualitative malate oxidation assay. Although clones of the gene for malate dehydrogenase (EC 1.1.1.37) are also selected by this method, we could isolate the malic enzyme gene from the Bacillus strain.

MATERIALS AND METHODS AND RESULTS’

Nucleotide Sequence of Malic Enzyme Gene-The nucleo- tide sequence of the gene that encodes the thermostable malic enzyme of B. stearothermophilus and its flanking region was identified by dideoxynucleotide chain termination sequencing in M13mp18 and M13mp19 vectors and is presented in Fig. 5. A 1434-base pair open reading frame starting at ATG (position 119) and ending at TAA (position 1552) was found; the open reading frame is big enough to code for a 478-amino acid residue peptide of 51,536 daltons. The N-terminal se- quence deduced for this open reading frame is identical to the sequence of the N-terminal region of the B. stearother- mophilus malic enzyme purified to homogeneity (Met- Ala-Leu-Pro-Gly-Gly-Ala-Ala-Met-Asn-Ile-Thr-Ile-Arg-Leu- Gln). The number of amino acid residues/subunit predicted from this open reading frame agreed well with that calculated from the result of amino acid analysis of the purified protein (numbers in parentheses): Lys, 31 (28.0); His, 6 (5.6); Trp, 1 (0); Arg, 24 (22.7); Asp + Asn, 52 (46.4); Thr, 24 (22.1); Ser, 19 (17.2); Glu + Glu, 38 (40.0); Pro, 22 (21.0); Gly, 37 (35.2); Ala, 53 (52.2); Cys, 7 (0); Val, 47 (46.2); Met, 10 (8.4); Ile, 49 (44.8); Leu, 33 (33.0); Tyr, 11 (10.4); and Phe, 14 (13.5). From these observations, we conclude that this open reading frame encodes the malic enzyme.

There is a ribosome-binding sequence complementary to

Portions of this paper (including “Materials and Methods,” part of “Results,” Figs. 1-5 and Tables I-V) are presented in the miniprint at the end of this paper. The abbreviations used are: IPTG, isopropyl- @-D-thiogalactoside; HPLC, high performance liquid chromatogra- phy; X-gal, 5-bromo-3-indolyl-~-~-galactoside; Mops, 4-morpholine- propanesulfonic acid; SDS, sodium dodecyl sulfate; kbp, kilobase pair(s); kat, katals.Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

3200

Thermostable Malic Enzyme from B. stearothermophilus 3201

the 3'-end of 16 S rRNA of E. coli, ~'-oHAUUCCUCCA- CUAG-5' (25), Bacillus subtilis, ~'-~HUCUUUCCUCCA- CAAG-5' (26), and B. stearothermophilus, ~'-HoAUCU- UUCCUCCACUAG-5' (27), 9 base pairs upstream from the initiation codon (Fig. 5). Probable promoter sequences, the -35 and -10 regions, are found 90 (TTGTCT) and 67 (TAA- CAT) base pairs upstream of the initiation codon, respec- tively, which have four of six bases in common with the consensus sequences recognized by E. coli RNA polymerase (TTGACA, -35 region; TATAAT, -10 region) (28). Analysis of the DNA sequence downstream from the coding region shows a short hairpin-loop structure (AG = -26.0 kcal/mol) (29) followed by A + T-rich sequences that seemed to be a termination signal.

DISCUSSION

The B. stearothermophilus malic enzyme obtained in this work is similar to E. coli (NAD-linked enzyme) and S. solfa- taricus enzymes in the molecular weight of the subunit (MI 50,000) (2, 3) but is different from the E. coli (NADP-linked enzyme; M, 62,0000) ( l ) , C. thermocellum (NADP-linked en- zyme; M, (40,000) (4), and murine (NADP-linked enzyme; M, 64,000) (10) enzymes. Plant NAD-linked malic enzymes (So- lanum tuberosum and Crassula argentea) are composed of two nonidentical subunits with molecular weights of 61,000 and 55,000 (7).

The thermal stability of the B. stearothermophilus malic enzyme is far higher than that of the enzymes isolated from mesophilic organisms (30). The stability is similar to that of the enzyme from another thermophilic bacterium, C. ther- mocellum (4), but is lower than that from S. solfataricus (half- life of the enzyme activity of 85 "C = 5 h) (3).

The B. stearothermophilus enzyme shows typical Michaelis- Menten behavior (between 0.5 and 10 mM malate) at 50 "C and pH 8.0. Unlike the calf mitochondrial enzyme, no sig- moidicity was observed.

Amino acid sequences of the murine malic enzyme were identified from the nucleotide sequences of cDNAs. No overall homology was observed between the B. stearothermophilus and murine enzymes. However, Harr plot analysis of the two sequences revealed several short homologous regions (Figs. 6 and 7A). One region ('51Val-Leu-Gly-Leu-Gly-Asp-Ile-Gly'5~) seems to constitute the NAD-binding domain since the se- quence is homologous not only to the sequence of the murine enzyme (15611e-Leu-Gly-Leu-Gly-Asp-Leu-Gly'63), but also to the sequence of the consensus NAD-binding site proposed by Wierenga et al. (31). Twenty-four amino acids out of 44 amino acids were also identical in another region (amino acids 233- 276) of the B. stearothermophilus enzyme and the region

B s t M E 1 5 1 m m m m - b Z i I E d ~ l Z G l l 5 8 Murine ME 15611e - Leu m 6 3 sMOH Ell Thr Ala Ala E/ Gln Ala17

mMDH @I IEi lm Ala Ser D Gly m3 LDH-A 2md Val [a Val - Ala Val m3 GluDH 24WE Gln Phe - Asn Va l W356 LAOH 1 9 m Phe lam - Q Gly Val m 0 4 GAPDH 5 I l e Asn l a Phe - Arg I D m l 2

FIG. 6. Alignments of sequences showing homology of B. stearotherrnophilus malic enzyme (Bst ME) with portions of NAD-binding regions of several dehydrogenases including murine malic enzyme (Murine ME), two isoenzymes of mouse malate dehydrogenases (cytosol enzyme (sMDH) and mito- chondrial enzyme (mMDH), pig lactate dehydrogenase (LDH- A ) , bovine glutamate dehydrogenase (GluDH), horse liver alcohol dehydrogenase (LADH), and B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The conserved amino acids are boxed.

(E) Bst M E VFHODQHCTAVVLLACLLNALKI V O K K L E D I W L T W C - A A C I A

M u r i n e ME TFNDDl QGTASVAVAGLLAALRI TKNKLSWNLFOCAC€AALCI A

2 3 3 276

" * * * * * * * * * t. . *t f * t *

265 31 0

FIG. 7. A, Diagonal comparison of B. stearothermophilus malic enzyme (Bst M E ) and murine malic enzyme (Murine ME). Dots were plotted where more than 6 out of 15 amino acids were identical between the two sequences. B, comparison of a portion of amino acid sequences of B. stearothermophilus and murine malic enzymes. Amino acids conserved between the two enzymes are indicated by asterisks.

(amino acids 265-310) of the murine enzyme (Fig. 7B). Thus, these malic enzymes seem to be evolutionarily related.

There was no significant homology between the malic en- zyme and various malate dehydrogenases (32-35) except for short homologous regions at the probable NAD-binding re- gions (Fig. 6). Irrespective of these observations, comparative structural analysis between the malic enzyme and malate dehydrogenases would provide us with important information about the structure-function relationship of these enzymes. Moreover, site-directed mutagenesis of the cloned malic en- zyme gene will also be effective for analyzing this relationship.

The nucleotide sequence of the B. stearothermophilus en- zyme also showed, upstream of the coding region, a ribosome- binding sequence and a probable promoter sequence that is similar to the consensus promoter sequence of E. coli. These sequences seem to contribute to the efficient expression of the gene in E. coli.

Acknowledgments-We thank H. Nakajima (Unitika Co. Ltd.) for providing B. stearotherrnophilus cells, K. Yamaguchi and T Fujio (Kyowa Hakko Co. Ltd.) for analyzing the amino acid sequence of the N-terminal regions of the malic enzyme, and F. Sakiyama (Insti- tute for Protein Research, Osaka University) for amino acid analysis of the enzyme.

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3202 Thermostable Malic Enzyme from B. stearothermophilus

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. .

499-560 Tanase, S., Kuramitsu, S., Kagamiyama, H., and Morino, Y.

StrA BUPE endl shcE15 hsdR4, F'traD36 DIOAB lac19 lacZnnl5) was used a9 the recipient for transformation (12). The plasmid pUC12 was used as the vector

EDntaining 10 g Of Tryptone, 5 g of NyCl, 5 g of yeast extract, and 1 g of plasmid for cloning experiments (13). E. strains were grown on LE medium

glucose per 1, pH 7.2, to a density Of 2 - 3 x 109 cells/ml. When necessary, ampicillin I50 pg/Ol) was added to the medium.

-

=me= and Chemicals ~estriction endonucleases (%I, w a r , Q R I , %RV. %cII, U d I l I ,

=I. =I, "1 SmaI and X I ) , and T4 DNA ligase were obtained from myobo Co. Ltd. (Osaka). k A G a s a generous gift from Kojin CO. Ltd. (Tokyo). NADP l a 8 obtained from the Oriental Yeast co. Ltd. (Tokyo). Other chemicals used were guaranteed grade reagent- fro. Nakarai Chemical Co. Ltd. (UYotol.

Preparation of DNA. Liqation, and Transformation Ch~omosomal DNA of & stearothermo hilus was prepared by the method of

Salt0 and Miura (14). Plasmid DNAs rere;repared from & Strains by the methods Of Eirnboim and mly (15) . followed by purification hy cesium chloride- ethidium bromide density gradient centrlfuqation. Ligation -as done as described previously 116). E. coli strains were transformed by the method of Cohen et al. (17). TransformFntGre seleted on LE medium containing 12 g Of agar, 4 T 1 9 of X-gal. 24 ag of IPS, and 50 m g of ampicillin per liter.

Detection of E. coli hansfo-nt Cells Producinq Thematable *alate DehydrOgeMse

described previously (18) with the following modifications. Ampicillin- Cells producing thermostable malate dehydrogenase were detected as

resistant transformants were traneferred to a paper filter, and the filter was soaked at 30°C for 20 rain in buffer I ( 2 0 01M Tris-HCI. pH 1.5, 10 m l l EDTA) containing 2 mg/ml of lysozyme. after the filter was washed with buffer 1, the cells were lysed by incubating the filter in 1 S Triton X-100 for 5 min at

dehydrogenase of & ,,U hoat cells. Positive clones were selected by 30°C. Then the fllter was Incuhatedat6SoCfor 30 mintoinactivatethe malate

incubating the filter at 30°C for 10 m i n in 20 mn Tris-8C1, pH 7.5, 1 0 0 mn D,L-malate 0 .5 mu 3-(4'.5'-dimethylthia.ol-2.yl)-2,5-diphe~ylt~t~~~~li~~ bromide, 0.; RM 5-ethylphenazinium ethylsulfate, and 50 pM NAD.

Gel EleatrO OreQiS Agarose~gel eleCtrophOze%ls of DNA was done as described preYiously (16).

~ ~ l y a c r ~ l a a i d ~ gel electrophoreses of protein were done ~n 7.5 3 polyacrylamide gel at pH 8 by the method of mVis (191. After electrophoresis. protein bands were stained with Coomassie brllliant blue R250 and then destained by 20 8 methanol/l % acetic acid solution. Malate dehydrogenase activity was detected by incubating the gel at r o o m temperature with 50 n M TriS-HC1 buffer, pH 1.5, containing 10 mg phenazine methasulfate 50 m g NAD, 25 mg nitrobluetatrarDlivs, and r n m m m a l a t e in 50 ml of mixture L v the method of rrenderson (20) wlth a

oxalacetate, 0.1 m U NADH, 20 mn KCI, and 5mU MgCl (for oxalacetate teductlonl; (for malate oxidation); 3 0 On potassium phosphate buffer IpB 1.5). 0.5 rnn

and 100 mn naps buffer IpH 6.1). 10 m H sodi& pyruvate, 100 ml4 sodium carbonat= 0.1 m l l NADH. and 5 rll MgCl (for pyzuvate reductian). Decarboxyla- ting act;tities Of oxalacetate were !ssayed at 30°C in a reaction mixture containing 100 mM potaSsiUm acetate (PA 4.6). 10 IN oxalacetate, 20 011 KCI. and 1 n W Y n C L : the CO, a a s evolved vas measured by a manometer. Proteins were

l290.0001 The &lecular weight of the subunit of the enzyme was measured by SDS-

polyacrylamide gel electrophorenis with the following standard proteins: trvosin inhibitor (20,100). carbonic anhydrase 17o.nnnl. ovalhumin 143.0001.

. .

KCTA, and disrupted by il French press ( 6 0 0 kg/cn2). The supernatant Obtained by centrifugation (9 000 x q, 10 mi") was used 8 8 the crude enzyme solution. To inactivate the mllate dehydrogenase of &a host cells. the solution was incubated at 60°C for 40 min. after removal of precipitate by centrifugation

The enzyme was eluted by a l inear gradient (0 to 0.5 M I of sodium chlorlde in a total volume of 2 1. The active fractions were pooled and concentrated by ultrafiltration with a UP-20 ultramembrane IAdvantsch Co.Ltd., Tokyo). The concentrated sample ( 5 m 1 ) was purified by high performance gel filtration chromatography (TSK-G3000SW) equilibrated with 50 mM potassiu. phosphate buffer. OB 7. containing 0.1 ll Of sodium chloride. The purified samples thus

The purifled sample was hydrolyzed at 1 1 0 OC in 6 M HC1 containing 0.2 U phenol for 24. 48, and 72 h. The amino acids were measured by an amino acid aMlyrer (Ritachi Co. Ltd. m o d e l 835). The amino acid sequence of the protein was identified by an autoiated gas-phase sequencer (Applled Biosystem Co. Ltd.. model 170A).

at a constant voltage (1900 VI

RESULTS

Thermostable Malic Enzyme from B. stearothermophilus 3203

5' c-"-.---" 3' - " Taq I

"-cI ___w -*

SsuSA I 1 "

.. - 2wbps

sequencing strateqy cor the m11s Enzm Gena r1g. 1. ~..trictio. m p Of pKD1 and

fragment containing the malic enzyme qene of & StearothermoDhiluS. The DNA Plasmid pMD1 is a hybrld plasmid consisting of pUCl2 and the 1.6 kbp -1

sequence was found by the didsoxynueleotida chain tsrmlnatlon method. The arrows lndlcata the direction and extent of the DNA squenclnq. The position of thsmallcenryme gene Is shown by an open box.

harboring pMD1 still retalned 120 nkatlmg of malate dehydrogenase actislty After 1nCllb.tio. at 60% for 40 min, the w l l extract of E. m l i 511103

(malate oxidation), but that of the homt cell lost almost all of the activity 0.5 nkatlmql. Cells hsrborlnq pMD1 had two active band. on polyscrylamlde

slab gel electrophoresis Irelatin, mobility: 0.2 and 0.4) IPIq 2 slot 6) The

enzyme migratlng as a slower band wsa stable up to 65OC (slots 7-91. Thls fa*ter-migratlng band disappeared sftar 40 mi" at 6OoC (aiot'l2). but the

moblllty was the M m e as that of the major band shown by original UK788 cells lrslstiva mobllity: 0.21 (slots 1-51. Traneformation of &- JM105 wlth pMD1 resulted in the prcdvctlon of a tharmoatable enxyma wlth malate dehydro- genase actlvity. These results indicate that pMD1 encodes the gene of the enzyme that corresponds to the major band of the a atearOthemODhilYs.

harboring pMD1 still retalned 120 nkatlmg of malate dehydrogenase actislty After 1nCllb.tio. at 60% for 40 min, the w l l extract of E. m l i 511103

(malate oxidation), but that of the homt cell lost almost all of the activity 0.5 nkatlmql. Cells hsrborlnq pMD1 had two active band. on polyscrylamlde

slab gel electrophoresis Irelatin, mobility: 0.2 and 0.4) IPIq 2 slot 6) The

enzyme migratlng as a slower band wsa stable up to 65OC (slots 7-91. Thls fa*ter-migratlng band disappeared sftar 40 mi" at 6OoC (aiot'l2). but the

moblllty was the M m e as that of the major band shown by original UK788 cells lrslstiva mobllity: 0.21 (slots 1-51. Traneformation of &- JM105 wlth pMD1 resulted in the prcdvctlon of a tharmoatable enxyma wlth malate dehydro- genase actlvity. These results indicate that pMD1 encodes the gene of the enzyme that corresponds to the major band of the a atearOthemODhilYs.

1 2 3 4 5 6 7 6 9 0 1 1 8 1 3

JM103 harboring pMD1 Islots 6-10), and JM103 (.lots 1 1 - 1 3 T w z The Cell extract. Of mtm~~Otb8rmODhll~. OK788 (Slots 1-5). E Coli

electrophoresed on a polyacrylamide slab (pH 8 , nom-denatured) gel, and than the bands of malate dshydrogeMses were detected by tetrazolium-llnked mpllnq aystsm (see *Materials and Methods"). In (10.11 experiments, the cell extracts of S. stearothermophilun, & - harboring pMD1, and &a (0.25 mg each)

heat treatment: slots 2 and 7; 55%: slots 3, 8, and 12; 6OoC: ilo;m I , 9, and for 5 m l n , and the resulting supernatant were applied. slots 1 6 and 11; no

13: 65%: slots 5 and lo; 70%.

were incvbated at var'lou. temperature for 20 m l " and centrifuged at 10,000 x p

Pluiflcatlon md o.ller.1 alaraet.rimtion O f tho oras Rodvet m characterize the enzyme produced by pMD1, we purlfled tha enzyme from

E. e011 harborlng pllD1. Table I summarizes a typlcsl purlflcstion procedure. Seventy percent of the .alata-oxldatlon aCtlvltle?l were retained after hest-

cally decreased to 0.5 a of the untreated sample. These results suggest that treatment of the crude extracts, but Oxalacatate reduction aCtlvItIes drasti-

the thermostable malate-oxldatlon actlvity Is due to the .all= enzyme from StearothermoDhlluS, and that the thermolabile Oxa1aEetate"eduCtion activity Is &e to the enzy.es of the e host cella.

"

Purification of & steamthamch ilu. m11c B1.m Table I

f- * mrboring plml

Purlflution Protein lctisity (pkat)

.tep 1.91 O.idatiOn reduction reddUction Malate Old1.Cetate Pyruvate

Call extract 5080 neat treatant 3730 Dm-Sephader A50 135 EPIC(TSK-C3OOOSWl 57

552 267

440 70

1.23 21.7 383 0.60 15.7 303 < 0.02 11.7

rig. 3. SDB-Polyacryl"ide Gel Electmphoresis of Malic Enzyme

electrophoresis Usin9 the toflowlnq standard proteins: trypsin inhibitor The purified enzyme (1 9) was analyzed by SDS-polyacrylamide gel

120,100). carbonic anhydrase 130,000~. ovalbumin (43,0001. bovine serum albumin (67,0001, and phosphorylase b 197.4001. The protein bands were detested rlth Cmmassle brllliant blue.

- mZm 6.0 5.2 4.8 3.7 t mzpe 10.0 26.0 21.0 17.3

polyacrylamide q.1 eleotrophoresis using standard proteins (Fig. 31. The The molecular weight of the subunit of the enzyme was 48,000 by SDS-

molecular weight of the native enzyme estimated by high performance gel flltratia chromatography (G3OOOSWl was 200,000. These results indicate that the M t l v e enzyme l a a tetramer composed of four identlcal subunits.

As shown in Fig. 4, the pR Opt1.a for malsts-oxldatlon and pyruvate-reduc- t i a are 8.0 and 6.0, respectively. For testing the pR stabillty of the enzyme,

described In the legend to Flg. 4. Remalnlng activltles were assayed at 50°C by the enzyme labout 10 pglml) was kept at 4 c for 24 h In 0.1 M buffers as

adding d small portion of the sample 1100 p1) to the 3 m1 of the standard reactlon nlltvre for the malate oxidation assav. Then. results show that the

pH

Fig. 4. Dapsndence of mllc Knrm Mtirlty on pE

buffer. One-tenth M each of the follalnq buffers rere msed; acetate buffer (pa 4.0-5.61 1 0 1 . phosphate buffer (pa 6.0-8.01 (01. Ris-EC1 buffer (8).0-9.0) (01, qlycine-NaOH buffer (pA 9.0 -10.6) ( A I , caroMte buffer (pE 9.2-10.81

.alate oxidstlon and pyruvate reduction. rsspectlrely. The a n r m sctirltlss 1.1 and Mope buffer IPS 6.1-7.5) (A). The solid line and broken line a s n

pLatl.9 for malate-orldatlon, at pR 8.0; 0.21 pkatllq for pyruvsta-reduction, rere arpresaed as percentages of the actlvltlss tmard the hlghest value. (5.4

mzm. rere a.eayed in the standsrd -&Ion .l*"re except for tha

at pn 6.0).

ahownl. Poc sraminlnq the thermwtabillty of the enzyme, the enzyme In 0.1 w RH Optimal tempsratu-e for the dehydrosenase activlty was 5S°C (data w t

potassimm phosphate buffer, pa 7.5. was Incubated at various temperatures for

untreated sample. Elqhty percent of the enzyme activities were stlll retained 20 m l n , then the residual sctivltior were assayed and compared wlth the

after incubation at 60%.

enzyme actlvlty. The addition of 0.05 mM EDTA to the reaction mixture caused Table 111 smmmarlzes data showing the effect. of divalent cations on the

complete loss of enzyme sstirity 0.5 nkstlmql, indicating that the enzyme rwlr'os divalent cations for activity. Addition of 0.5 mM Mn2+ restored the activity to the level obtained by the standard malate-oxidation assay described In "natsrlals and Methods. 15.4 pkatlmq, see Table 11. Mq2*, Co2* and Zn2*

also dependent on the monovalent cations (Table IV ) . NR4+ and K t enhance the activity about 5 times. ua+ and Li' have w effect on the enhancement. Similar activation by divalent and monovalent cations w a ~ also reported for several malic encymes (3, 41.

partiany restored tha activity, but -2' was bsr-1~ effssti-. me activity I.

eltraEtS, wlth 0 yield,of 30 8 based on the activity of malate oxldatlon. The The thermostable malic enzyme was purified 100-fold over the cruds

absorption cwffieisnt E:.:,. of the lyophilized snryme was 4.9. The enzyme appeared to be homogeneous, yielding a single protein h n d by SDS-polyacryl- amide gel elestrophorasls (Pig. 3). The final sample had a malate oxldstion actlvlty of 5 4 lratlmg (at 50°Cl. When 0.5 mM Of NADP was added instead of 0.5 mM NAD. the a&ty decreased to 6 (0.32 pkatlng). The purlfisd enzyme had pyruvate reduction actlvlty In carbonate buffer (0.21 pkatlmq at pA 6 . 0 , F l q . I ) , but has no Oxalacetate reductLon activity 0.29 nkatlmq, Table I). Though spontan-(I decarboxylation of the oxalacetate was observed, espedally at lor pH, Table I1 clearly shows that the enzyme ala0 catalyzes the decarboxylstlon of oxalacetste In the absence of NADIP) (0.87 pkatlmg at pA 4.61. These data

enrrmel (ECl.l.l.381. lndlcste that the enzyme Is malate dehydrogenase ldecarborylatlng) (mallc

3204 Thermostable Malic Enzyme from B. stearothermophilus

Ug2* 0.5 51

0.05 2 n?+ 0.5

0.05 0.6

' 0.1

m2* 0.5 56

0.05 ' 0.1

NOnS < 0.1

~ ~~

CAtiw. (.*I Ennryw act1rity Cati- l u l l n=ya a0ti.ity In0 addition - 1) (DO addition = 1 I

I* 20 7.1 n.+ 20 1 .o t 2.8 1 1.0

ua,* 20 4.7 Li' 20 1 .o 1 5.7 1 1 .o

Thermostable Malic Enzyme from B. stearothermopkilus 3205

4

0 0,

W i- t-

:I % <

V V

e <

V < i-

0 i-

V 0

V i- i- t- c

t- W :I t- V w < i- 4

< < V

c < W

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W < < < V < V w i- U

v m

v m C Y < < < < < n e& < < <n

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V I . V d v < w < r3h O R w r ( 0-4 w v 00 v * V a l u c w * <i- v < v u

V 0 I- 0 0 V i-

e e i- i- i- e V V 0 < < w 0 W 0

i- <

i- < i- i-

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t- i- V V W V < 0 0 V < 0 V V < < 0 W e 0 < V W e < < w 0

G

0 <

- < x c i - o O N 0 i-

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0 < 0 <

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V I W < < < W < < w i- < < < < < < V < o < - I i- < i- i- < 0

's < i- c i- < W i- < 0 < e < 0 V V

i- <

C d Cr( 4 v u

< h i - a V I 4

<-l w * < I . 0 V a0 v Y N V I.u t-VI-v<- i - c w m < n V r l < < 0-2 < * vr( v c i - a <i- w > < a v h <r( or( 00 o w i - u 0 0 t - . - I V & <n v a <VI w * < h v r O h v * <-l < e w r ) < h w w hi- O h < & d-4 i - m v u w e d w w w w cr( v *

w * urn v c w * <i- v < uaa < * w * v c v < <i- v a <yI <VI < h w < <-I u * w - v u i - m c l m w w

c 4 kr( u u u

<-4 < n v V I . w a t- w w w < v V L : < d t- v = o w a o i - <i- o w < < ul-i- u m < W < N V - l N W

rl < h 9 <-l