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Biochimica et Biophysica A

Molecular analysis of the nitrile catabolism operon of the

thermophile Bacillus pallidus RAPc8B

Rory A. Cameron, Muhammed Sayed, Don A. Cowan*

Advanced Research Centre for Applied Microbiology, Department of Biotechnology, University of the Western Cape, Bellville 7535, Cape Town, South Africa

Received 20 August 2004; received in revised form 24 March 2005; accepted 28 March 2005

Available online 2 May 2005

Abstract

The gene cluster containing the nitrile hydratase (NHase) and amidase genes of a moderate thermophile, B. pallidus RAPc8 has been

cloned and sequenced. The (5.9 kb) section of cloned DNA contained eight complete open reading frames, encoding (in order), amidase

(belonging to the nitrilase related aliphatic amidase family), nitrile hydratase h and a subunits (of the cobalt containing class), a 122-

amino acid accessory protein, designated P14K, a homologue of the 2Fe-2S class of ferredoxins and three putative proteins with distinct

homology to the cobalt uptake proteins cbiM, cbiN and cbiQ of the S. typhimurium LT2 cobalamin biosynthesis pathway. The amidase

and nitrile hydratase genes were subcloned and inducibly expressed in Escherichia coli, to levels of approximately 37 U/mg and 49 U/

mg, respectively, without the co-expression of additional flanking genes. However, co-expression of P14K with the NHase structural

genes significantly enhanced the specific activity of the recombinant NHase. This is the first description of an accessory protein

involved in thermostable NHase expression. Modelling of the P14K protein structure has suggested that this protein functions as a

subunit-specific chaperone, aiding in the folding of the NHase a subunit prior to a-h subunit association and the formation of a2h2

NHase holoenzyme.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Nitrile hydratase; Bacillus; Thermostable; P14k; Chaperone

1. Introduction

Nitrile hydratases (NHase) EC 4.2.1.84 catalyse the

conversion of nitriles to their corresponding amides. The

physiological role of the enzyme has recently been linked to

the microbial metabolism of aldoximes, forming the second

stage in a three-step pathway involving aldoxime dehydra-

tase, NHase and amidase in the synthesis of carboxylic acids

from their corresponding aldoximes [1]. A recent study of

the distribution of aldoxime dehydratase in microorganisms

0304-4165/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbagen.2005.03.019

Abbreviations: NHase, nitrile hydratasei The nucleotide sequences reported in this paper are available from the

DDBJ/GenBankTM/EMBL Data Bank under the accession number

AY184492.

* Corresponding author. Tel.: +27 21 959 2083; fax: +27 21 959 3505.

E-mail address: dcowan@uwc.ac.za (D.A. Cowan).

URL: www.biotechnology.uwc.ac.za.

demonstrated that all active strains tested exhibited a nitrile

or aldoxime co-induced nitrile degrading activity [2].

Nitrile degrading enzymes, and in particular NHases,

have been the subject of considerable academic and

commercial interest for over two decades, largely due to

their potential as industrial biocatalysts. Currently, immobi-

lised NHase producing microorganisms are used in the

kilotonne-level synthesis of acrylamide (Mitsubishi-Rayon

Chemical Co., Japan) [3], nicotinamide (niancinamide,

vitamin B3) (Lonza Guangzhou Fine Chemicals, China)

[4] and 5-cyanovaleramide (5-CVAM), a starting material

for the synthesis of a DuPont herbicide, azafenidin [4].

NHase genes have been cloned from various Rhodococ-

cus species [5–11], Pseudomonas chlororaphis [12],

Pseudomonas putida [13] and from the thermophiles

Pseudonocardia thermophila [14], Bacillus smithii [15]

and Bacillus sp. BR449 [16]. In addition, the recent

completion of several microbial genomes has revealed

cta 1725 (2005) 35 – 46

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–4636

probable NHase genes in two legume symbionts: Meso-

rhizobium loti (NCBI Microbial Genomes Annotation

Project) and Sinorhizobium meliloti [17]. In each case, the

a and h subunits were coded within two separate, adjacent

open reading frames. There is a high degree of similarity

between the NHase sequences, and a number of strictly

conserved regions. In particular, all share the characteristic

cofactor-binding motif CXLCSC where X is serine in iron-

containing enzymes and threonine in the cobalt-containing

enzymes [18]. With the exception of the NHase operons of

Rhodococcus rhodochrous J1, all the enzymes described

above are co-expressed with an amidase, the gene for which

is generally positioned approximately 100 bp upstream from

the NHase genes.

Initial attempts at recombinant expression of active

NHase were largely unsuccessful, resulting in the produc-

tion of insoluble and inactive inclusion bodies [10,11,19].

Heterologous expression of active NHase was later facili-

tated through the development of host–vector systems in R.

rhodochrous strains for the expression of recombinant

Rhodococcus NHases [20] and through the discovery that

the co-expression of ‘‘activator proteins’’ from genes

flanking the NHase genes significantly enhanced active

over-expression [12,21]. Despite high sequence identity

between Co-type and Fe-type NHases, their corresponding

activators share no homology whatsoever: those associated

with the Fe-type NHases of P. chlororaphis [12] and

Rhodococcus sp. N771 [22] are 47 kDa proteins with

homology to the ATP dependant iron transporter MagA,

while the Co-type activators are small (¨14 kDa) proteins

with homology to NHase h subunit. Mutational studies of

NHase activator of Rhodococcus sp. N-771 have identified

a proposed metal-binding motif CXCC essential for NHase

activation furthermore, an interaction between the activator

and NHase was also observed (irrespective of mutations),

indicating a role as an Fe-type metallochaperone [23]. The

14-kDa Co-type NHase activators, however, do not contain

known metal-binding motifs. Nevertheless, studies of the P.

putida P14K protein have shown that it is expressed at very

low level, leading to the proposition that it serves as a

chaperone or acts in a catalytic capacity [24]. Moreover, it

was shown that the incorporation of cobalt into the active

site is not a passive process. Thus, the chaperone/catalyst

hypothesis seems to indicate that the 14-kDa NHase

activators (h homologues) are involved in the integration

of cobalt into the active site.

The NHases of R. rhodochrous J1 [10], Rhodococcus sp.

ACV2 [25], Rhodococcus sp. N-774 [21], Rhodococcus sp.

N-771 [22], P. chlororaphis B23 [12] and P. putida 5B [24]

have now been expressed as active enzymes in E. coli or R.

rhodochrous strains in the presence of their respective

activators. The expression of Bacillus sp. BR449 NHase in

E. coli [16] was unique in that only the NHase structural

genes were required for expression of activity: expression of

the P12K protein of Bacillus sp. BR449 was not detected

when the p12k gene was included in expression constructs,

neither did the gene seem to affect NHase expression or

activity.

A thermostable nitrile hydratase and its associated

amidase were purified to homogeneity from a thermophilic

aerobe, identified using molecular phylogenetic methods as

Bacillus pallidus [26]. This thermostable Co-dependant

enzyme was found to possess a broad specificity for linear,

cyclic and branched aliphatic substrates, for dinitriles and

for heteroaromatic nitriles but to be inactive on aromatic

nitriles [27]. Here, we report the detailed analysis of the

NHase operon and its flanking regions and employ

sequenced-based modeling as a means of probing enzyme

structure–function relationships.

2. Materials and methods

2.1. Strains, plasmids and culture conditions

B. pallidus sp RAPc8 was previously isolated in our

laboratory [26]. E. coli TOP10 (Invitrogen) and E. coli

JM107 [28] were hosts for cloned PCR products and

genomic DNA, respectively; E. coli BL21 (DE3) was used

as a host for over-expression. pCR2.1 (Invitrogen) and

pUC18 were used for the cloning of PCR products and

genomic DNA, respectively, and the expression vector

pET21a (+) (Novagen) was used for over-expression. All

E. coli cells were grown in LB medium at 37 -C; selectivemedium was supplemented with ampicillin or carbenicillin

(100 Ag/ml�1). B. pallidus RAPc8 cells were grown in 2�LB medium at 60 -C.

2.2. Cloning of the B. smithii RAPc8 NHase locus

Chromosomal DNA isolation, restriction digests, electro-

phoresis and Southern blotting methods were conducted as

previously described [29]. Plasmid DNAwas prepared from

5 ml overnight cultures using the QIAprep Spin Miniprep

kit (Qiagen) according to the manufacturer’s instructions.

All PCR products were purified using the QIAquick PCR

purification kit (Qiagen).

Degenerate PCR primers, 5V-GTTGTNAARGCCTGG-ANCGATCC-3V (NHF) and 5V-RTARCABGARCAYAR-NGTRCARAC-3V (NHR), where B=G+T, N=A+C+G+T,

R=A+G, Y=C+T, was designed based on conserved regions

in alignments of amino acid sequences of the NHase alpha

subunit family. PCR reactions using B. pallidus RAPc8

genomic DNA as a template contained: 2.5 U Taq DNA

polymerase (Roche Molecular Biochemicals, Germany), 1�standard PCR buffer containing 1.5 mM MgCl2, 1.2 AMeach primer and 5–10 ng template DNA, 0.2 mM dNTPs in

a 50-Al reaction volume. The reaction conditions were: 5

min at 94 -C, followed by 25 cycles of 30 s at 94 -C, 30 s at

55 -C and 30 s at 72 -C and a final extension at 72 -C for 7

min. A PCR product within the expected size range (¨170

bp) was amplified and cloned into pCR2.1 (to produce

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–46 37

pF2I). The insert of pF2I was sequenced and found to

exhibit significant similarity to the equivalent fragments of

previously reported NHase a subunit genes (data not

shown).

The amplified fragment was used as a probe for

Southern hybridization against genomic B. pallidus RAPc8

DNA digested with several common restriction enzymes.

Labeling and detection procedures were performed using

the DIG DNA labeling and detection kit (Roche Molecular

Biochemicals) according to the manufacturer’s method.

The PCR-amplified a subunit regions were located in a

¨4.5 kb BamHI fragment and a ¨3.2 kb XhoI fragment of

B. pallidus genomic DNA. Based on this information, two

E. coli size-enriched libraries were constructed, containing

either 4–5 kb BamHI or 3–4 kb XhoI digested B. pallidus

RAPc8 genomic DNA cloned into a pUC18 plasmid.

These libraries were screened by colony hybridization

using the same probes. A plasmid, designated pNHB4,

isolated from a positive colony in the BamHI library was

sequenced and found to contain the full a subunit and

approximately half of the h subunit. Despite repeated

attempts to screen the XhoI library, none of the screened

colonies hybridized with either probe under high strin-

gency conditions, whilst a high degree of unspecific

hybridization was obtained when the stringency conditions

were relaxed.

Primers 5V-GTTCATTGGTCCCAGCTCAT-3V (C8IN1)

and 5V-CATGAAGGCGGTAAACCAAG-3V (C8IN2),

designed against the NHase a subunit sequence in pNHB4,

were then used to isolate the remainder of the upstream

sequence of the NHase operon by inverse PCR. PCR

reactions using 100 ng re-ligated XhoI fragments of B.

pallidus RAPc8 DNA was used as a template in PCR

reactions containing: 1.5 U Pfu DNA polymerase (Promega

Corp.), 1� Pfu buffer containing 2 mM MgSO4, 1 AM each

primer 0.2 mM dNTPs in a 50-Al reaction volume. The

reaction conditions were: 5 min at 94 -C, followed by 25

cycles of 30 s at 94 -C, 30 s at 58 -C and 3 min at 72 -C and

a final extension at 72 -C for 7 min. The reactions were

performed in duplicate in order to verify upon sequencing

that no errors had been introduced during amplification. The

products of these reactions, 3.1 kb fragments containing the

entire amidase and NHase h subunit genes and a portion of

the NHase a subunit gene, were cloned into pCR2.1 to

produce pINX2 and pINX4.

2.3. DNA sequencing and sequence analysis

All DNA sequences were determined at Oswel DNA

(University of Southampton), with an Applied Biosystems

377 DNA sequencer and a DNA sequencing kit. Sequence

analysis manipulation and annotation were performed on

DNAstar (DNASTAR Inc.) and Gene Construction Kit

(Textco BioSoftware, Inc.) and BioEdit programs (North

Carolina State University). Nucleotide and protein similarity

searches were the GenBank and SwissProt databases were

performed with the BLAST algorithm on the NCBI server

[30]. Sequence alignments were performed using the

CLUSTAL W [31] facility on the EMBL server. Nucleic

acid secondary structure predictions were performed using

MFOLD [32]. Primers were designed either manually or

with the aid of the online Primer3 tool (Whitehead Institute/

MIT Center for Genome Research). The nucleotide

sequence data have been deposited in GenBank under the

accession number AY184492.

2.4. Subcloning, expression and preparation of cell-free

extracts

Primers incorporating specific restriction sites were

designed for directional subcloning into the expression vector

pET21a: 5V-GCTCATATGAGACACGGGGATATTTC-3V(AMD3, NdeI); 5V-GCACGGCCGTATTCCAGAATTACA-CACC-3V (AMD4, EagI); 5V-GCTCATATGAACGGTATT-CATGATGTTGG-3V (NHOP1, NdeI); 5V-GCAGCGGCCGQCATTAATAAAAAACCTCATCTCG-3V (NHOP2, NotI);

5V-GCAGCGGCCGCCTAACCTACCGTAACTTTAGG-3V(NHOP3, NotI) and 5V-GCTCATATGAAAAGTTGTGA-GAATCAACC-3V (NHOP4, NdeI). Using primer pairs

AMD3/AMD4, NHOP1/NHOP3, NHOP1/NHOP2 and

NHOP4/NHOP2, four separate PCR products, containing

the amidase, NHase, NHase-P14K and P14K genes,

respectively, were amplified from genomic B. pallidus

RAPc8 genomic DNA using Pfu polymerase. Each product

was tailed with single 3V A overhangs and cloned into

pCR2.1 as described in the technical manual (Invitrogen

Corp.), to produce pNH22 (amidase), pNH51 (NHaseha),pNH46 (NHasehaP14K) and pNH32 (P14K). The inserts of

these plasmids were excised by means of the NdeI and NotI

(pNH51, pNH46 and pNH32) or NdeI and EagI (pNH22)

sites engineered into the primers and subcloned into the

respective sites in pET21a. The resulting plasmids: pNH223

(amidase), pNH512 (NHaseha), pNH461 (NHasehaP14K)and pNH321 (P14K) were introduced into E. coli

BL21(DE3)-competent cells.

Recombinant protein expression was induced with

isopropyl-1-thio-h-d-galactopyranoside to 0.4 mM final

concentration. Cells were harvested 16 h after induction,

washed and resuspended in 1/20th culture volume of 50 mM

potassium phosphate buffer, pH 7.2. The cells were

disrupted by sonication (Bandelin Sonopuls), and the extract

clarified by centrifugation (20 000�g, 20 min).

2.5. Enzyme assays

Amidase and NHase activity was measured using cell-

free enzyme solutions in 500 Al reactions containing 50 mM

acetamide or acetonitrile as substrate. All assays were

carried out at 50 -C. Conversion of nitriles or amides to the

corresponding acids was assayed through the release of

ammonia by a modification of the phenol-hypochlorite

ammonia detection method as previously described [26].

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–4638

When measuring NHase activity, the reaction mixture was

supplemented with an excess of B. pallidus RAPc8 amidase

(partially purified by heat-treatment–data not shown). One

unit of enzyme activity was described as the amount of

enzyme that catalysed the release of 1 Amol of NH3 per

minute under standard assay conditions.

Protein concentrations were determined using the Bio-

Rad Bradford assay, using bovine serum albumin as a

protein standard.

2.6. N-terminal amino acid sequencing

Cell-free samples of NHase were electrophoresed in a

15% SDS PAGE gels and transferred to PVDF membranes

(Roche). Following transfer, the membranes were stained

with Coomassie blue, destained and air-dried. The NHase a

and h subunit bands were cut out and sent for Edman

sequencing at PNAC (University of Cambridge).

2.7. Model building and macromolecular docking

The threading program FUGUE [33] was used for the

identification of structural homologues and structure

prediction. Alignment outputs from FUGUE were sub-

sequently used for model building using the external

program Modeller [34]. Structural modeling of B. pallidus

RAPc8 NHase was based on the structure P. thermophila

NHase (PDB code 1ire [35]). This was the most suitable

template based on the high degree of sequence conserva-

tion (59% and 40% identity for a and h subunits,

respectively). N-terminal residues 1–9 and C-terminal

residues 212–216 of the NHase a subunit were omitted

from the alignment used as these regions are very poorly

conserved amongst NHases. P. thermophila NHase hsubunit was also used as the template for modeling of

P14K. The sequence identity over the homologous region

(h subunit N-terminal ¨100 amino acids) is only 18%,

giving rise to concern for the accuracy of alignment

prediction. However, confidence in the alignment was

supported by the observation that the positions of helices

and loops predicted for P14K using the PHD program

[36] corresponded with those P. thermophila NHase hsubunit.

Stereochemical analysis of the structures was performed

using PROCHECK [37], the final models display good

geometry, with less than 1% of residues in the disallowed

region, although the P14K model does have less residues

(73%) in the most favoured region than would be desired for

a highly accurate model.

Docking calculations for the modeling of the interaction

between NHase and P14K were performed using the

Fourier correlation-based program GRAMM [38]. The

coordinates used for the docking procedure were those

obtained from the previous modeling steps, for NHase,

only the coordinates of the a subunit were used. The

program parameters were set as suggested for low-

resolution docking [38]. The 10 lowest-energy scoring

orientations were retained for screening. All models and

docked structures were viewed and presented as figures

using PyMOL [39].

3. Results

3.1. Nitrile hydratase operon structure

The nitrile hydratase gene cluster (Fig. 1) was partially

isolated from a plasmid pool containing BamHI fragments

of B. pallidus RAPc8 chromosomal DNA. The positive

plasmid (pNHB4) isolated by colony screening contained

about 4.4 kb of chromosomal B. pallidus RAPc8 DNA in

the BamHI site of pUC18. However, sequencing revealed

that although the clone contained the entire NHase a subunit

gene, only 346 bp (approximately half) of the a subunit

gene had been cloned. Despite repeated attempts to clone

the 5V region of the NHase operon by colony hybridization,

no colony reacting to the probe was identified. Subse-

quently, primers C8IN1 and C8IN2 (designed against the

sequence of the pNHB4) were amplified by inverse PCR:

products within the expected size range of approximately

2950 bp were cloned and sequenced. Assembling the

sequences of pNHB4, pINX2 and pINX4 allowed the

determination of the NHase coding sequence, including

the upstream and downstream-flanking sequences.

Translational analysis of the complete 6594 bp locus

revealed eight putative ORFs and a ninth, truncated at the 3VBamHI site of the pNHB4 insert. BLAST homology

searches identified these ORFs as: amidase; NHase h;NHase a; an ORF which translated to a 14-kDa protein with

homology to the Co-type NHase Factivator_ proteins as wellas the NHase h subunit; a 2Fe-2S type ferredoxin

homologue and homologues of the cbiN, cbiM, cbi Q and

cbiO cobalamin biosynthesis genes. All ORFs had the same

polarity, started with an ATG codon and terminated with a

TAA codon with the exception of NHase a (TAG) and cbiQ

(TGA). Potential Shine–Dalgarno ribosome binding

sequences (S.D.) were identified 6–9 nucleotides upstream

of the start codons of each potential gene with the exception

of cbiQ. The consensus S.D. sequence was (A/G)G(A/G)(A/

G)G(A/G). The overall GC content of the cloned DNA was

40.2%.

The first ORF encoded a 348-amino acid protein (MW

38.6 kDa), with 100% identity with the amidase of Bacillus

sp. BR449 [16]. However, due to six silent nucleotide

substitutions, the DNA sequences shared a slightly lower

identity (99%).

The NHase genes, encoded in h–a order, were located

127 bp downstream of the stop codon of the amidase. The

NHase h subunit gene encoded a protein of 229 amino

acid (26.5 kDa), the N-terminus of which corresponded

precisely to the previously determined N-terminus of the

purified NHase h subunit of B. pallidus RAPc8 [40]. The

Fig. 1. (A) Nitrile hydratase gene cluster of B. pallidus RAPc8, open reading frames are represented by closed arrows, bar=1000 nucleotides. (B) Coding sequence of the genes encoding amidase, NHase h and a

subunits and P14K. Putative ribosome binding sequences are underlined.

R.A.Camero

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al./Biochimica

etBiophysica

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1725(2005)35–46

39

Fig. 2. Expression of the amidase, NHase and P14K. All samples were

induced with 1 mM IPTG, protein expression was allowed to proceed for

16 h, approximately 10 Ag clarified cell extracts were electrophoresed on

15% SDS-PAGE gels. Lanes 1 and 9: molecular weight markers. Lanes 2–

8: E. coli BL21 (DE3) containing: 2: pET21a; 3: pNH223; 4: pNH461

(grown in 0.1 mM CoCl2); 5: pNH461 (grown in absence of CoCl2); 6:

pNH512 (grown in 0.1 mM CoCl2); 7: pNH512 (grown in absence of

CoCl2) and 8: pNH321. Labels: Amd—amidase, beta—NHase beta

subunit, alpha—NHase alpha subunit.

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–4640

entire sequence showed significant sequence identity (31–

98%) to known NHase h subunits, particularly that of

Bacillus sp. BR449 (98% identity). The NHase a subunit

gene, encoding a protein of 216 amino acids (24.6 kDa)

and containing the motif CTLCSCY (residues 116–121),

characteristic of cobalt binding enzymes [18], was found

25 bp further downstream. Although the sequence dis-

played high similarity to many a NHase sequences (46–

98% identity), it showed absolutely no similarity to the

N-terminal sequence deduced from the purified protein

[40].

The fourth ORF was found only 13 bp further down-

stream and encoded a 14.6-kDa protein of 122 amino acids.

BLAST searches indicated that it had significant similarities

(Fig. 3) to P12K of Bacillus sp. BR449, NhhG [9] and NhlE

[41] of Rhodococcus J1, P14K of P. putida, and hypo-

thetical proteins of S. meliloti and M. loti (81%, 33%, 29%,

12%, 25% and 22%, respectively), all of which are

associated, at least by proximity with the NHase genes of

their respective organisms. Interestingly, with the exception

of P. putida P14K, these proteins also have a low but

significant similarity (15–30%) to the N-terminus of their

related NHase h subunits.

A strong (DG =�14.6 kcal mol�1) hairpin structure that

may serve as a U-independent transcription termination

signal [42] was located 11 bp downstream of the termination

codon of the p14k gene. The absence of significant structures

elsewhere in the sequence and the proximity of the NHase a

and h and p14k genes suggests that these are all co-

transcribed as a single mRNA which most likely also

contains the amidase sequence. This would be in accordance

with the suggestions in the literature regarding the related

NHase operons of Rhodococcus sp. R312 [25], Rhodococcus

erythropolis [7], P. chlororaphis [12] and P. putida 5B [43].

269 bp downstream from the genes of this proposed

nitrile hydratase operon, ORFs 5–9 were organised in a

manner that suggested that they may be transcribed in a

single polycistronic mRNA so as to form part of a separate

operon. Each gene has significant homology to genes

involved in cobalt uptake and linked to cobalamin biosyn-

thesis pathways.

The first gene of this putative operon, encoded a 119

amino acid (13.8 kDa) protein with similarity to the 2Fe–2S

class of ferredoxins [44] and had a 48% amino acid identity

with a hypothetical ferredoxin of B. halodurans [45] and

39% with a ferredoxin homologue of B. megaterium,

designated cbiW, the first in the cbi (cobalamin biosyn-

thesis) operon of the latter organism [46].

ORFs 6–9 encoded putative peptides of 252 aa (24.7

kDa), 100 aa (11 kDa), 215 aa (24.7 kDa) and 159 aa, the

highest scoring BLAST hit in each case being with putative

cobalt uptake proteins of the putative cobalamin biosyn-

thesis pathway of Clostridium perfringens [47]: ORF6—

56% identity with cbiM; ORF7—45% identity with cbiN;

ORF8—34% identity with cbiQ; ORF9—52% identity over

159 aa with cbiO.

3.2. Over expression of the amidase, nitrile hydratase and

P14K genes in E. coli

Four constructs were used in the investigation of

recombinant NHase/amidase production in E. coli BL21

(DE3). The cloned genes were expressed from the tightly

regulated IPTG inducible T7lac promoter of pET21a. As

shown in Fig. 2, both NHase (pNH512 and pNH461) and

amidase (pNH223) were expressed at high levels as soluble

protein (estimated from SDS-PAGE to be at least 30% in

each case) and required no additives other than IPTG. P14K

was not detectable on SDS-PAGE gels in either the soluble

or insoluble cell fractions when co-expressed with NHase

(pNH461) and was expressed as insoluble inclusion bodies

when expressed alone (pNH321) (data not shown). The N-

terminal amino acid sequencing of the a and h NHase

subunits confirmed the sequences deduced from nucleotide

sequence except the N-terminus methionine was not

observed in the a subunit, probably as a result of post-

translational processing.

The activities of the crude extracts of the recombinant

proteins are detailed in Table 1. To investigate the effect of

the addition of cobalt ions to the growth medium, a series of

expression experiments were performed with varying

concentrations of CoCl2. However, initial experiments

revealed that the addition of cobalt inhibited the growth of

the E. coli host (data not shown). For this reason, cobalt was

only added 15 min prior to induction.

Expression from pNH512 in the absence of Co2+ resulted

in the production of a large quantity of soluble apoenzyme

(Fig. 2) with very low activity. Very similar activities were

Table 1

Specific activities of the crude extracts of the recombinant enzymes on 50 mM acetamide (amidase) and 50 mM acetonitrile (NHase)

Sample Co2+ in growth media Specific Activity (U/mg)

Crude extract Heat treatment only Cobalt activation

Amidase (pNH223) – 40 – –

NHase ha (pNH512) 0.1 mM 50 97 136

NHase ha (pNH512) – 0.5 – 200

NHase ha + P14K (pNH461) 0.1 mM 1260 2094 1955

NHase ha + P14K (pNH461) – 0 – 75

Cobalt activation and heat treatment experiments were conducted by incubation of dilute (0.5 mg/ml total protein) enzyme samples at 50 -C for 30 min, in the

presence of 5 AM CoCl2 where relevant.

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–46 41

observed when cobalt was added to cultures at concen-

trations of 0.1 and 0.5 mM. A maximum level of specific

activity on acetonitrile of 50 U/mgT4 U/mg was achieved

by supplementation with 0.1 mM CoCl2. When cells

harboring pNH461 were grown in the absence of cobalt,

NHase was produced but no activity was detected. How-

ever, when supplemented with 0.1 mM CoCl2, highly active

NHase was produced, with a specific activity of 1260 U/mg:

more than 25 times the activity of NHase alone.

3.3. NHase cobalt activation

When grown in the absence of cobalt, both BR449 and

RAPc8 NHases are expressed as inactive but folded

apoenzyme. Bacillus sp. BR449 NHase apoenzyme,

expressed in the absence of cobalt in the growth media,

can be Factivated_ by incubating crude NHase extracts at 50

-C in the presence of 5 AM Co2+: crude extracts of activated

samples were approximately seven times more active than

the same extracts grown in the presence of cobalt [48]. This

experiment was repeated with B. pallidus RAPc8 NHase

(Table 1). Incubation of NHase apoenzyme with 5AM Co2+

resulted in a four-fold increase in activity to 200 U/mg

whilst the activation procedure had a less marked effect on

samples grown in cobalt-supplemented media and virtually

no effect on those co-expressed with P14K. The 40–50%

rise in activity for the latter samples can be attributed to the

partial purification of the enzyme by heat denaturation of the

native E. coli proteins.

4. Discussion

The nitrile hydratase and flanking genes of B. pallidus

RAPc8 were cloned and expressed in E. coli. The 5V portionof the described locus was recalcitrant to cloning by

conventional library production, forcing us to use PCR

techniques to determine this section of the NHase operon. It

is possible that the amidase or NHase genes contained

within the ¨3.2 kb XhoI fragment may have been expressed

from their own promoters, producing recombinant protein

toxic to E. coli. However, although NHase has often been

expressed as inactive inclusion bodies in E. coli [11,19], the

literature contains no reports of either NHase or amidase

being toxic—even at high levels in E. coli. Thus, we cannot

currently offer a reasonable explanation for the resistance to

cloning.

When the predicted N-terminal sequence of the two

NHase subunits was compared with those reported by

Pereira et al., no sequence homology was found between the

two a subunit N-terminals. Sequencing of the recombinant

a subunit N-terminus confirmed the sequence predicted by

computational translation. It is possible that, as with

Rhodococcus sp. J1, B. pallidus RAPc8 produces more

than one NHase, or, that the N-terminal sequence previously

reported [40] was incorrect. Since the observation of only

one hybridizing fragment suggests that only one set of

NHase genes is present on the chromosome, it is probable

that the previously reported N-terminal data are erroneous.

The remarkably high sequence identity between this

operon and that of Bacillus sp. BR449 [16] suggests that

they originate from very closely related Bacillus strains. A

comparison of the 16S rDNA of B. pallidus and the

corresponding 485 bp from Bacillus sp. BR449 [49]

revealed a 99.79% sequence identity (data not shown),

supporting this conclusion. Though it is likely that Bacillus

sp. BR449 was isolated from geothermal soil or samples

similar to that from which RAPc8 was isolated, its precise

origin has not been reported.

Previous studies on a number of NHase-producing

Rhodococcus species have demonstrated that related organ-

isms contain NHase genes with high sequence identities

[7,11,21]: comparisons of the NHases of R. erythropolis and

Rhodococcus sp. N-774, which share 94.7% and 96.2%

amino acid sequence identity for the a and h subunits,

respectively, demonstrated that a very limited number of

amino acid changes can significantly change NHase activity

and substrate specificity [7]. Therefore, whilst it is clear that

the enzymes produced by the two Bacillus species are

closely related, it would be reasonable to expect variations

in catalytic behaviour.

4.1. Transcript production

The organisation of the amidase, NHase a and h and

P12K genes, the similarity of their Shine–Dalgarno sequen-

ces and the presence of a stem loop structure just downstream

suggests that they are transcribed as a single polycistronic

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–4642

mRNA. It is thought that a similar transcriptional organ-

isation is used by the NHase operons of Rhodococcus sp.

N-774 [21], Rhodococcus sp. N-771 [22], R. erythropolis

[7], P. chlororaphis B23 [12] and P. putida 5B [43]. In a

mutant strain of Rhodococcus sp. R312, Rhodococcus sp.

ACV2, this has been conclusively demonstrated by northern

blot analysis [25]. Based on the spacing between the NHase

and amidase genes of Bacillus sp. BR449 and the significant

heterologous NHase expression in reverse orientation to the

vector promoter, Kim and Oriel proposed that a promoter

upstream to the NHase genes may facilitate their expression

independently of the amidase. While no promoter elements

were detected within the cloned DNA of B. pallidus RAPc8,

confirmation of either hypothesis can only be irrefutably

demonstrated by transcript analysis.

B. pallidus RAPc8 amidase belongs to the nitrilase-

related amidase family, conversely, all other previously

described NHase-associated amidases (with the exception of

Bacillus sp. BR449) belong to the aspartic proteinase class

[50]. While previous literature suggests that the closely

related NHase operons of a diverse range of microbes have

been acquired by horizontal gene transfer [40,51], the

anomaly presented by the amidases of these two Bacillus

species indicates that pre-existing genes were also utilized in

the assembly of the NHase operon.

4.2. Significance of the cbi homologues

Translated BLAST searches with the sequences of ORFs

5–9 revealed that each had significant homology to a gene

involved an ABC cobalt transport system. More specifically,

with the cobalt uptake genes of the cobalamin (vitamin B12)

biosynthesis operons of either Bacillus megaterium [46] or

Salmonella typhimurium [52]. Although gene clustering is

evident in both B. megaterium and S. typhimurium, the gene

orders are quite different, with a number of the individual

genes being unique to one organism. Interestingly, each

RAPc8 homologue was unique to either B. megaterium or S.

typhimurium, suggesting that they may not be crucial genes

in cobalamin biosynthesis. ORF 5 shared 39% sequence

identity with the cbiW gene product of B. megaterium, a

ferredoxin homologue that is suggested to act as an oxido-

reductant [46].

ORFs 6–9 showed significant homology (between 54

and 20% amino acid sequence identity), to the gene

products of (in order): cbiM; cbiN; cbiQ; and cbiO of S.

typhimurium (which are the only genes unique to that

organism c.f. B. megaterium), the latter three are known to

be membrane associated proteins involved in cobalt uptake

[52]. Whilst the homologous genes in B. pallidus RAPc8

are found in the same order, unlike their S. typhimurium

counterparts (which often overlap), there is significant

spacing between the genes and only one potentially func-

tionally associated gene found upstream.

Further investigation, including cloning of the sequence

downstream of the 4.5-kb BamHI fragment, is clearly

required before grounded predictions can be made as to the

function of these genes. However, the proximity of the

genes to an operon producing a cobalt-containing enzyme,

and the absence of further cobalamin biosynthesis genes

upstream does suggest that they are involved in uptake and

supply of cobalt to the NHase. This view is further

supported by the fact that iron and cobalt transport proteins

have been found associated with other NHase operons.

NhlF of the R. rhodochrous J1 L-NHase gene cluster, a

member of the nickel/cobalt transport family [53], has been

shown to confer cobalt uptake on Rhodococcus and E. coli

hosts and its expression significantly enhances L-NHase

activity in Rhodococcus transformants in cobalt limiting

conditions [41]. A 1188-bp ORF found in the operons of

Rhodococcus sp. N-774 and N771 encodes a protein that

bears distinct homology to an ATP-dependant iron-trans-

porter, magA of Magnetospirillum sp. AMB-1 and,

interestingly, to cobW (distinct from cbiW) of P. denitri-

ficans. This ORF has found to be important for high-level

recombinant NHase expression [21,22].

If indeed it is the case that these genes can mediate the

high affinity transport of cobalt for supply to NHase, then it

may be that heterologous co-expression of these genes with

the NHase genes would enable the expression of a high

level of NHase activity without the need to supplement the

growth media with CoCl2. This is the first example of an

NHase-associated ABC transport system which further

corroborates our argument that the NHase gene cluster has

been assembled from pre-existing genes.

4.3. Expression studies

Both the NHase and amidase enzymes were actively

expressed without the need for co-expression of additional

genes, in keeping with previous findings [26] that the

expression of nitrile degrading activity by the native B.

pallidus RAPc8 was constitutive. The results obtained in

this work were also consistent with previous findings that

presence of cobalt is required for the expression of NHase

activity rather than protein production [9,10,16,19]. It is

clear from the cobalt-heat activation experiments that, even

in the presence of cobalt, when expressed alone, much of the

NHase is present as inactive NHase apoenzyme. Higher

levels of activity are attained by incubating the NHase

apoenzyme with cobalt at elevated temperature. However,

the highest levels of activity overall were detected when

NHase was co-expressed with P14K. Conversely, coex-

pression of the Bacillus sp. BR449 P12K protein with

NHase did not seem to affect NHase expression or activity

nor was the gene product detected when the p12k gene was

included in expression constructs [16]. Given the 100%

conservation of amino acid sequence between P12K and

P14K, this lack of activity is most likely due to the

truncation of the P12K protein. Whether this was due to a

sequencing error or mutation that caused the loss of function

has yet to be determined. Although the requirement for the

Fig. 3. ClustalW alignment of the 14-kDa activator proteins of B. pallidus RAPc8, Bacillus sp. BR449, R. rhodochrous J1 H-NHase (J1Nhhg), L-NHase

(J1Nhle) and P. putida 5B. Key: *, conserved amongst all NHase beta subunits; +, conserved amongst all Co-type NHase beta subunits; – , highly conserved

between activator and beta subunits.

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–46 43

expression of flanking genes and the production of

accessory proteins for recombinant NHase activity has been

described for mesophilic organisms [27], this has not

previously been reported for the thermophiles.

4.4. Significance of the 14-kDa protein

P14K has significant sequence similarities with the beta

subunit homologues P12K of Bacillus sp. BR449; NhhG,

NhlE, of the high and low molecular weight NHase operons

of R. rhodochrous J1 (Fig. 3); and, to a limited extent P14K

of P. putida 5B—all associated with the activation of Co-

type NHases. All are encoded immediately downstream of

their respective NHase genes and appear to be part of a

polycistronic operon.

Whilst the precise function of the p14k gene products is

unknown, it has been found that their presence is essential

for functional heterologous expression of R. rhodochrous J1

and P. putida 5B NHases in E. coli [9,10,24,41]. Studies of

the P. putida P14K protein have shown that it is expressed at

a very low level, leading Wu et al. [24] to postulate that it

serves as a chaperone or acts in a catalytic capacity. When

B. pallidus RAPc8 P14K was co-expressed with NHase, no

Fig. 4. Four possible mechanisms for the formation of NHase holoenzyme: (A) ahCo bind uniformly (C) Co binds to folded alpha subunit, subsequently h subunit b

binding of Co, P14K subsequently replaced by h subunit.

corresponding 14 kDa band was detected on SDS-PAGE

gels, despite a marked effect on NHase activity, indicating

either that this protein is expressed at low levels, or is

rapidly degraded following NHase folding. In each case, it

has been shown that cobalt is required for activity rather

than for expression or correct folding and that its incorpo-

ration into the enzyme is not passive. The chaperon/catalyst

hypothesis has been used to suggest that the P14K proteins

are involved in the integration of cobalt into the active site

[54].

We propose that the P14K protein acts specifically as a

subunit-specific catalyst. Fig. 4 indicates four possible

routes for the biogenesis of NHase and incorporation of

cobalt. The ability to partially activate NHase in vitro to

higher levels than NHase expressed in the presence of cobalt

(Table 1) suggests that, in the absence of accessory proteins,

NHase apoenzyme is formed prior to the incorporation of

cobalt ions, as in situation (a). NHase is most active when

co-expressed with p14k in the presence of cobalt. However,

in vitro cobalt activation of the same construct expressed in

the absence of cobalt results in substantially lower levels of

activity, indicating P14K cannot activate NHase subsequent

to apoenzyme formation. Based on these results and the

apoenzyme forms first followed by the binding of Co cofactor (B) a, h and

inds to form holoenzyme (D) a subunit stabilized by P14K to allow for the

Fig. 5. Superimposition of the model of P14K on the model of the h subunit

of B. pallidus RAPc8 NHase. The beta subunit is shown in green, P14K in

magenta. The figure was generated using PyMol.

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–4644

distinct homology between P14K and the h subunit (¨25%

sequence identity over 100 amino acids), we propose

situation (d) as the most probable route of biogenesis of

fully active NHase in vivo. We suggest that the partially

folded a subunit is stabilized during the binding of the

cobalt ion through an interaction with P14K, which is

subsequently substituted for the h subunit to produce the

fully folded NHase holoenzyme. In order to illustrate this

hypothesis, sequence-based homology models of B. pallidus

Fig. 6. (A) Model of P14K (magenta) docked to NHase alpha subunit (yellow). (B

shown in yellow, beta in magenta. In each model, the cobalt atom is represented

numbering) are shown as sticks in green and that of the conserved arginine (R59

RAPc8 NHase and P14K (Figs. 5 and 6) were constructed,

using the structure of the P. thermophila NHase as a

template. According to this proposition, the NHase a-P14K

complex is necessarily a transient one and the ability of

current docking algorithms based on rigid-body searches to

reliably predict such complexes is limited [55]. Never-

theless, as a means of illustration, our folding models show

clearly that the P14K NHase activator is capable of binding

to the alpha-subunit with similar geometry to the h subunit

of the native NHase. Moreover, upon analysis of residues

conserved between the NHase h subunits and Co-type

NHase activators (Fig. 3), it becomes apparent that the

majority correspond to the aromatic residues that stabilize

the active site and cofactor binding region of previously

described NHase structures through an extensive hydrogen-

bond network [56], and possibly through aromatic–aro-

matic interactions [57]. Examples of such residues are P14K

Y82 and Y83, (NHase hY72 and hY73), highlighted in Fig.

5, the corresponding tyrosines of Rhodococcus sp. N-771

NHase add significantly to the stability of the structure

through H-bonds with each other, a single hydration water

molecule and aS113 (N-771 numbering) [56]. P14K R59

(NHase hR56) is also strictly conserved throughout all

NHases: the equivalent arginine has been shown to form a

hydrogen bond with the oxygen atoms of both modified

cysteine residues on the alpha subunit that serve as ligands

to the metal cofactor in both Co-type and Fe-type NHase

[35,56]. The conservation of the sequence, overall archi-

tecture and geometry of these important residues between

P14K and B. pallidus RAPc8 NHase thus strongly suggests

a similar binding mechanism, supporting our hypothesis for

NHase activation.

) Model of the ah dimer of B. pallidus RAPc8 NHase, the alpha subunit is

in red. The side chains of the conserved tyrosines (Y82 and Y83–P14K

–P14K numbering) in orange.

R.A. Cameron et al. / Biochimica et Biophysica Acta 1725 (2005) 35–46 45

Given the degree of homology between the B. pallidus

RAPc8 P14K protein and the homologues from other

organisms (22–82% amino acid identity), we further

propose that the proposed mechanism for chaperone-like

stabilization of folding intermediates is a generic mechanism

for most if not all cobalt-type nitrile hydratases.

Acknowledgements

The authors gratefully acknowledge the following for

financial support: the BBSRC (UK), the NRF (SA) and the

Royal Society/NRF Structural Biology program (UK/SA).

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