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Originally published In Press as doi:10.1074/jbc.M405686200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 10, 8660-8667, March 11, 2005
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Nitrile Pathway Involving Acyl-CoA Synthetase

OVERALL METABOLIC GENE ORGANIZATION AND PURIFICATION AND CHARACTERIZATION OF THE ENZYME*

Yoshiteru Hashimoto{ddagger}, Hideaki Hosaka{ddagger}, Ken-Ichi Oinuma, Masahiko Goda, Hiroki Higashibata, and Michihiko Kobayashi§

From the Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan

Received for publication, May 21, 2004 , and in revised form, December 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Two open reading frames (nhpS and acsA) were identified immediately downstream of the previously described Pseudomonas chlororaphis B23 nitrile hydratase (NHase) gene cluster (encoding aldoxime dehydratase, amidase, the two NHase subunits, and an uncharacterized protein). The amino acid sequence deduced from acsA shows similarity to that of acyl-CoA synthetase (AcsA). The acsA gene product expressed in Escherichia coli showed acyl-CoA synthetase activity toward butyric acid and CoA as substrates, with butyryl-CoA being synthesized. From the E. coli transformant, AcsA was purified to homogeneity and characterized. The quality of the recombinant protein was verified by the NH2-terminal amino acid sequence and the results of matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The apparent Km values for butyric acid, CoA, and ATP were 0.32 ± 0.04, 0.37 ± 0.02, and 0.22 ± 0.02 mM, respectively. AcsA was shown to be a short-chain acyl-CoA synthetase, according to the catalytic efficiencies (kcat/Km) for various acids. The substrate specificity of AcsA was similar to those of aldoxime dehydratase, NHase, and amidase, the genes of which coexist in the same orientation in the gene cluster. P. chlororaphis B23 grew when cultured in a medium containing butyraldoxime as the sole carbon and nitrogen source. The activities of aldoxime dehydratase, NHase, and amidase were detected together with that of acyl-CoA synthetase under the culture conditions used. Moreover, on culture in a medium containing butyric acid as the sole carbon source, acyl-CoA synthetase activity was also detected. Together with the adjacent locations of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase genes, these findings suggest that the four enzymes are sequentially correlated with one another in vivo to utilize butyraldoxime as a carbon and nitrogen source. This is the first report of an overall "nitrile pathway" (aldoxime->nitrile->amide->acid->acyl-CoA) comprising these enzymes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have extensively studied the biological metabolism of toxic compounds (which have a triple bond between carbon and nitrogen) such as nitriles (R-C{cjs0809}N) (1–5) and isonitriles (R-N{cjs0809}C) (6–8). The microbial degradation of nitriles proceeds through two different enzymatic pathways (9–11): (i) nitrilase catalyzes the hydrolysis of nitriles into acids (R-C({cjs0808}O)-OH) and ammonia (12–14); and (ii) nitrile hydratase (NHase)1 catalyzes the hydration of nitriles to amides (R-C({cjs0808}O)-NH2) (15–18), which are subsequently hydrolyzed to acids and ammonia by amidase (19–21). These enzymes have received much attention in applied fields (2, 9, 22, 23) as well as academic ones (14, 24–28). One of the fruits of our application-oriented nitrile studies is the current industrial production of acrylamide and nicotinamide using NHase of an actinomycete, Rhodococcus rhodochrous J1 (1, 10). On the other hand, NHase of Pseudomonas chlororaphis B23 (29), which was previously used as a catalyst for acrylamide manufacture (10, 22), is now used for the production of 5-cyanovaleramide, a herbicide intermediate, at the industrial level (30). Because the amount of NHase produced comprises >50% of the total soluble proteins in P. chlororaphis B23 when this strain is cultured in the presence of methacrylamide (31), there must be a very interesting regulation mechanism for the enzyme expression (9). We have already cloned the NHase gene from this strain, and we discovered the existence of a gene cluster including the NHase and amidase genes (32). Very recently, we also initially discovered that the aldoxime dehydratase gene (oxdA) is an additional member of this gene cluster and that OxdA synthesizes nitriles from the corresponding aldoximes (R-CH{cjs0808}N-OH) (33–35); OxdA has been approved as a new enzyme by Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (EC 4.99.1.5 [EC] ). Although we have clarified the nitrile-degradative and -synthetic mechanisms at the protein and gene levels, further metabolism of acids that are formed from the corresponding amides by amidase has never been found.

In the present study, we discovered two genes (nhpS and acsA) downstream of the above-mentioned NHase gene cluster in P. chlororaphis B23. We constructed an Escherichia coli transformant overexpressing AcsA, purified the AcsA, and determined its enzymological and physicochemical properties. Although various types of enzymes involved in acid metabolism in living organisms have been reported, we reveal here for the first time that AcsA plays an essential role in acid utilization in the nitrile-degradative pathway, resulting in elucidation of the whole "nitrile pathway."


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—All acyl-CoA compounds and acids were purchased from Sigma. DEAE-Sephacel, HiPrep 16/10 Q XL, and a low molecular weight standard kit were obtained from Amersham Biosciences. Standard proteins for high-performance gel filtration chromatography were obtained from Oriental Yeast (Tokyo, Japan). All other biochemicals were standard commercial preparations.

Plasmids, Strains, and Media—Plasmid pPCN4 carrying the genes encoding NHase and P47K on pUC19 (32) was used as a probe to clone the downstream region in P. chlororaphis B23. E. coli DH10B (Invitrogen) was used as the host for pUC plasmids (36). E. coli BL21-CodonPlus(DE3)-RIL (Novagen, Madison, WI) was used as the host for one plasmid, pET-24a(+) (Novagen), and its derivative, and it was also used for expression of the acyl-CoA synthetase gene (acsA). E. coli acetyl-CoA synthetase-deficient mutant strain AJW1781 (CGSC#8076; {Delta}acs-1::kan thr-1 leuB6(Am) {Delta}(codB-lacI)3 rpsL136 metF159(Am) thi-1) (37) and long-chain acyl-CoA synthetase-deficient mutant strain K27 (CGSC#5478; fadD88) (38) were obtained from the E. coli Genetic Stock Center, Yale University (New Haven, CT). E. coli transformants were grown in 2x YT medium (39), unless otherwise noted. The culture medium for P. chlororaphis B23 was composed of 1% sucrose, 0.5% (NH4)2SO4, 0.05% KH2PO4, 0.05% K2HPO4, 0.05% MgSO4·7H2O, and 0.001% FeSO4·7H2O (pH 7.0). Unless otherwise stated, sucrose and (NH4)2SO4 were used as carbon and nitrogen sources, respectively.

DNA Manipulations—Restriction endonucleases, DNA polymerase, and T4 DNA ligase were purchased from Toyobo Co., Ltd. (Osaka, Japan). Nucleotides were sequenced by the dideoxy-chain terminating method using an ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA). Unless otherwise stated, DNA manipulations were performed essentially as described by Maniatis et al. (39).

Expression and Purification of Recombinant Acyl-CoA Synthetase— The coding sequence of the enzyme was amplified by PCR with pPCN4 (as described below) as a template. The following two oligonucleotide primers were used: (i) a sense primer, 5'-GAATTCTAAGGAGGAATAGCATATGCGCGATTATGAACACGTTGTTG-3' containing an EcoRI recognition site (underlined), a ribosome binding site (bold), a TAG stop codon in-frame with the lacZ gene in pUC18 (italic), a NdeI recognition site (double underlined), and 25 nucleotides of acsA starting with the ATG start codon; and (ii) an antisense primer, 5'-GGATCCTTAACCAAGCGCCTGTTGCTTGGAC-3', containing a BamHI recognition site (underlined) and 25 nucleotides that are complementary to the 3'-end sequence of acsA ending with the TAA stop codon. The amplified DNA was subcloned into vector pUC18 and checked by DNA sequencing. The insert DNA was digested with NdeI and BamHI and then inserted into the respective sites of pET-24a(+). The resultant plasmid was designated as pET-acsA; in this construct, acsA was under the control of the T7 promoter. As for pUC-acsA construction, EcoRI was also used for digestion instead of NdeI, followed by insertion into the respective sites of pUC18. In this construct, acsA was under the control of the lac promoter.

E. coli BL21-CodonPlus(DE3)-RIL was transformed with pET-acsA, and the recombinant cells were used for the overproduction and purification of acyl-CoA synthetase. The transformed cells were incubated with reciprocal shaking at 37 °C in 25 ml of 2x YT medium containing 50 µg/ml kanamycin and 34 µg/ml chloramphenicol. After overnight cultivation, the entire culture was inoculated into 5 liters of the same medium, followed by incubation with shaking at 37 °C for 2 h. Isopropyl 1-thio-{beta}-D-galactopyranoside was then added to a final concentration of 0.1 mM to induce the T7 promoter, and further cultivation was carried out at 28 °C for 12 h.

All purification procedures were performed at 0–4 °C. Potassium phosphate buffer (KP buffer) (pH 7.5) containing 1 mM EDTA and 10% (w/v) glycerol was used throughout the purification, unless noted otherwise. Centrifugation was carried out for 40 min at 10,000 x g.

The cells were harvested by centrifugation, washed twice with 0.1 M KP buffer, and then disrupted by sonication (Insonator Model 201M; Kubota, Tokyo, Japan) to prepare a cell-free extract. Cell debris was removed by centrifugation. The resulting supernatant was applied to a DEAE-Sephacel column (4 x 16 cm) equilibrated with 0.1 M KP buffer. Protein was eluted from the column with 0.4 liter of 0.1 M KP buffer. The enzyme was collected as flow-through fractions, followed by dialysis against three changes of 5 liters of 10 mM KP buffer. After centrifugation, the enzyme solution was loaded on a HiPrep 16/10 Q XL column (20 ml) equilibrated with 10 mM KP buffer. The enzyme was eluted by increasing the ionic strength of KCl in a linear manner from 0 to 0.2 M in the same KP buffer. The active fractions were combined and dialyzed against 10 mM KP buffer without EDTA and glycerol and then centrifuged. The homogeneity of the purified protein was confirmed by SDS-PAGE.

Enzyme Assays—Acyl-CoA synthetase activity was measured by means of the following two assay systems. All of the reactions were performed under linear conditions with regard to protein (~1 µg/ml) and time (~4 min).

The standard assay A mixture comprised 200 mM Tris/HCl buffer (pH 7.5), 5 mM butyric acid, 8 mM CoA, 5 mM ATP, 8 mM MgCl2, 100 mM (NH4)2SO4, and an appropriate amount of enzyme (acyl-CoA synthetase), in a total volume of 150 µl. The reaction was started by the addition of the enzyme and carried out for 4 min at 35 °C. The reaction was stopped by adding 150 µl of methanol to the reaction mixture, and a supernatant was obtained by centrifugation (15,000 x g, 5 min). The amount of butyryl-CoA formed was determined by high pressure liquid chromatography with a Shimadzu LC-6A system (Kyoto, Japan) equipped with a Cosmosil 5C18-AR-II column (reversed-phase, 4.6 by 150 mm; Nacalai Tesque, Kyoto, Japan). The mobile-phase solvent systems were as follows: (i) solvent A, 220 mM KH2PO4-H3PO4 buffer (pH 4.0) with 0.05% (w/v) dithiothreitol; and (ii) solvent B, 98% methanol with 2% chloroform (v/v). Dithiothreitol was added to the solvent systems to prevent oxidation of CoA during the analyses. Chromatographic separation was performed at 40 °C at a flow rate 1.0 ml/min. The mobile-phase composition profile during each chromatography was divided into several linear segments, with each successive segment exhibiting an increased slope. The mobile-phase composition was 94% solvent A/6% solvent B at time 0, 92% solvent A/8% solvent B at time 3, 87% solvent A/13% solvent B at time 4.5, 80% solvent A/20% solvent B at time 6, and 55% solvent A/45% solvent B at time 9. After the final composition of 55% solvent A/45% solvent B had been held for 1.5 min from 9 to 10.5 min, the original conditions were reestablished by means of a reverse gradient to 94% solvent A/6% solvent B from 10.5 to 13 min. This composition was held for 2 min from 13 to 15 min, at which time the column was ready for injection of another sample. The amount of each acyl-CoA sample as a product was measured by monitoring the column effluent at 254 nm (retention time: butyryl-CoA, 13.6 min; acetyl-CoA, 10.8 min). This assay was used to routinely measure acyl-CoA synthetase activity during the purification procedure and to determine the temperature and pH optima of the enzyme.

In the case of standard assay B, acyl-CoA synthetase activity was assayed by measuring the acid-dependent formation of AMP with a slight modification (40). The reaction was carried out at 35 °C in a mixture (300 µl) containing 60 µmol of Tris/HCl buffer (pH 7.5), 1.5 µmol of butyric acid, 0.9 µmol of CoA, 0.9 µmol of ATP, 0.9 µmol of MgCl2, 30 µmol of (NH4)2SO4, 0.6 µmol of phosphoenol pyruvate, 0.12 µmol of NADH, 0.6 unit of myokinase, 0.7 unit of pyrvate kinase, 0.8 unit of lactate dehydrogenase, and an appropriate amount of enzyme. The reaction was started by the addition of the enzyme and carried out for 4 min. The reaction was stopped by adding 300 µl of methanol to the reaction mixture, followed by measurement of the decrease in absorbance at 340 nm. This assay was used to determine the specificity of the enzyme for various acids, with the exception of butyric acid, propionic acid, and acetic acid; the amounts of acyl-CoAs corresponding to the latter three acids were measured by means of standard assay A.

One unit of acyl-CoA synthetase activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol butyryl-CoA/min and 1 µmol AMP/min under the standard assay A and B conditions, respectively. Specific activity is expressed as units/mg protein. kcat values were calculated with an Mr of 60,210 for the AcsA monomer.

The assaying of other enzymes was performed as follows. Aldoxime dehydratase activity was measured anaerobically under reduced conditions (standard assay B) as described previously (33). NHase activity was assayed by the method reported previously (29). Amidase activity was measured by the same method as reported previously (21).

Molecular Mass Determination—The purified enzyme sample was applied to a Superose 12 HR10/30 column (Amersham Biosciences), which was attached to an ÄKTA purifier (Amersham Biosciences) and then eluted with 50 mM KP buffer containing 0.15 M KCl at a flow rate of 0.5 ml/min. A Superose 12 HR10/30 or Hiload Superdex 200 prep grade 120 ml column (Amersham Biosciences) or a TSK-GEL G3000SW column (Tosoh Co., Tokyo, Japan) was also used. The absorbance of the effluent was recorded at 280 nm. The molecular mass of the enzyme was calculated from the mobilities of the standard proteins glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), adenylate kinase (32 kDa), and cytochrome c (12.4 kDa).

The mass of the protein was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (Axima-CFR plus; Shimadzu). Samples were prepared by mixing the purified protein with an equal volume of a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile, and 1 µl of this solution was spotted onto the MALDI target plate. For mass analysis, external calibration was carried out using two protein standards (equine cytochrome c, average m/z 12,361.96; and bovine serum albumin, average m/z 66,430.09) provided by Sigma.

Analytical Methods—Protein concentrations were determined with a Nacalai Tesque protein assay kit, with bovine serum albumin as the standard. SDS-PAGE was performed in a 12% polyacrylamide slab gel according to the method of Laemmli (41), unless otherwise stated. The gel was stained with Coomassie Brilliant Blue R-250. The relative molecular mass of the enzyme was calculated from the mobilities of the marker proteins phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and {alpha}-lactalbumin (14.4 kDa).

The NH2-terminal amino acid sequences were determined with samples electroblotted onto a polyvinylidene difluoride membrane after SDS-PAGE using a Procise protein sequencer (Applied Biosystems).

Concentration of extracellular acetate was measured with an F-kit acetic acid (J. K. International Inc., Tokyo, Japan) according to the method recommended by the manufacturer.

Nucleotide Sequence Accession Number—The nucleotide sequence data reported in this study appear in the DDBJ/GenBankTM database under accession number AB125061 [GenBank] .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Nucleotide Sequence of the 3' Downstream Region of the NHase Gene—In previous studies (32, 33), we found the close location of five genes, which encode aldoxime dehydratase (OxdA), amidase, the {alpha}- and {beta}-subunits of NHase, and P47K (which has been renamed NhpC), in that order. Although we also detected the existence of another open reading frame (ORF) just downstream of nhpC on sequence analysis, this possible ORF (named OrfE previously) has never been obtained as a complete frame; its 3'-coding region was deleted. In order to obtain the complete ORF and to clarify the overall gene organization involved in the nitrile metabolic pathway of P. chlororaphis B23, we tried to clone the downstream regions of the genes obtained so far by the DNA-probing method with a 1.2-kb fragment, which was isolated from pPCN4 (32) digested with XhoI plus SalI, as a probe (Fig. 1). Southern hybridization with this probe against total DNA from P. chlororaphis B23 digested with XhoI revealed that it hybridized with a single fragment of about 5 kb (data not shown). This DNA fragment was separated by agarose gel electrophoresis, ligated with pUC18 digested with SalI, and then introduced into E. coli DH10B. Colony hybridization with this probe for screening ampicillin-resistant transformants containing the restriction fragment yielded pPCN41 (Fig. 1). Sequence analyses of the fragment showed that two inserts from pPCN41 and pPCN4 shared a common 1.2-kb XhoI-SalI region.



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  		FIG. 1.
Organization of the gene cluster involved in the nitrile pathway in P. chlororaphis B23. pPCN4 and pPCN41 indicate the regions cloned by the respective plasmids. The probe used in the experiment is shown (white box). Arrows indicate the position of each gene and the orientation of the coding sequences.

 
We determined the whole sequence of the XhoI-XhoI 4.8-kb fragment cloned and newly found two complete ORFs clustered with and in the same orientation as the structural genes of aldoxime dehydratase, amidase, subunits {alpha} and {beta} of NHase, and NhpC (DDBJ/GenBankTM accession number AB125061 [GenBank] ). The first ORF (previously named OrfE), which has been renamed nhpS, is 1293 nucleotides long and should encode a protein of 430 amino acids. nhpS showed significant amino acid sequence similarity with a hypothetical protein, Chromobacterium violaceum ATCC12472 (40% identity). The second ORF, named acsA, is 1638 nucleotides long and should encode a protein of 545 amino acids. The protein encoded by acsA showed the highest amino acid sequence similarity with FadD1 (70% identity) from Pseudomonas putida U (Ref. 42; GenBankTM accession no AF290950 [GenBank] ), whose mutant is unable to grow in a minimum medium containing an n-alkanoic acid or n-phenylalkanoic acid (with an acyl chain of longer than C4) as sole carbon source. AcsA is homologous to acetyl-CoA synthetase (31% identity) and acyl-CoA synthetase (25% identity) from E. coli (GenBankTM accession numbers U00006 [GenBank] and L02649 [GenBank] , respectively). In particular, two highly conserved sequence elements that comprise the ATP-AMP signature motif (common to all members of the adenylate-forming superfamily) (43, 44), are found in AcsA (residues 193–204 and 334–339). In the structure of PheA that belongs to a family of adenylate-forming enzymes, Lys517 is a part of the active site, contacting both the AMP ribose ring oxygen and a carboxylate oxygen of the substrate (45). Also, the equivalent lysine, Lys430, is conserved in AcsA. On the other hand, we have never observed the fatty acyl-CoA synthetase signiture motif, which is restricted but highly conserved among prokaryotic and eukaryotic fatty acid acyl-CoA synthetases and is proposed to compose a part of the fatty acid binding site (46), in AcsA. It is suggested that acsA encodes a kind of enzyme that ligates acid and CoA. nhpS and acsA are preceded by nucleotide sequences AGAGG and AGGTG, respectively, which we assume each serve as a ribosome-binding sequence. The distance between the TGA codon for NhpC and the ATG start codon for nhpS is 17 bp. The distance between the TAG codon for nhpS and the ATG start codon for acsA is 112 bp. It is possible that the seven genes (i.e. oxdA, amiA, nhpA, nhpB, nhpC, nhpS, and acsA) are co-transcribed in P. chlororaphis B23.

Identification and Purification of the Second ORF Product, AcsA—An expression plasmid (pET-acsA) for the second ORF was constructed and introduced into E. coli BL21-CodonPlus(DE3)-RIL. The recombinant protein was almost completely obtained as an insoluble form when the E. coli transformant was cultured at 37 °C. To increase the amount of the soluble enzyme form, various culture conditions were examined. Under the optimum conditions of 12 h at 28 °C, we succeeded in expressing the recombinant protein at a level corresponding to about 10% of the total amount of soluble protein (Fig. 2). Cell-free extracts prepared from the E. coli transformant carrying the ORF acted on butyric acid, ATP, and CoA as substrates, with butyryl-CoA being yielded. This finding demonstrated that this ORF encodes acyl-CoA synthetase. Thus, the gene was designated as acsA, as described above.



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  		FIG. 2.
SDS-PAGE of AcsA enzyme samples at various stages of purification. Protein bands were detected by staining with Coomassie Brilliant Blue. Lane M, marker proteins; lane 1, cell-free extracts (10 µg); lane 2, DEAE-Sephacel chromatography (1.8 µg); lane 3, HiPrep 16/10 Q XL chromatography (1.4 µg).

 
Interestingly, most of the AcsA activity was lost during the enzyme purification. Dialysis of the cell-free extracts caused 92.1% loss of the initial enzyme activity. We found that the loss of activity was reversible and that the activity was restored on the addition of salt to the assay mixture (5 mM (NH4)2SO4, 41%; 50 mM (NH4)2SO4, 80%; 100 mM (NH4)2SO4, 99%; 200 mM (NH4)2SO4, 101%; 300 mM (NH4)2SO4, 105%; 400 mM (NH4)2SO4, 99%; and 500 mM (NH4)2SO4, 94%, respectively), which allowed the characterization of this enzyme in various ways after purification. Therefore, we added (NH4)2SO4 to the standard enzyme assay mixture. AcsA was simply purified as described under "Experimental Procedures." The purity of AcsA was confirmed by migration of the protein as a single band corresponding to a molecular mass of ~58 kDa on SDS-PAGE (Fig. 2). The purified AcsA had an NH2-terminal sequence of MRDYEHVVESFD. This is the same as that deduced from the DNA sequence. To determine the molecular mass of AcsA, we subjected the purified enzyme to gel filtration on a Superose 12 HR10/30 column. A single peak was eluted from the Superose 12 HR10/30 column at a position corresponding to an apparent molecular mass of 46 kDa, as determined from the migration of markers of known molecular mass. This value is not similar to that obtained when the enzyme was denatured with SDS and then subjected to PAGE. It is also not consistent with the molecular mass predicted from the amino acid sequence (60,210 Da). Therefore, further determination was carried out by gel filtration on other columns, i.e. TSK-GEL G3000SW and Hiload Superdex 200 prep grade 120-ml columns. With these columns, the molecular mass of the purified enzyme was estimated to be 38 and 48 kDa, respectively. The mass of the protein was also confirmed by MALDI-TOF mass spectrometry. AcsA gave a molecular ion peak at m/z 60,276, suggesting that the COOH terminus of the protein was not truncated. Taken together, these data suggest that the active form of AcsA is a monomer. The purified AcsA showed specific activity of 6.98 units/mg (Table I). The UV-visible spectrum of the colorless enzyme showed an absorption maximum near 280 nm and no further absorbance at higher wavelengths (data not shown).


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  		TABLE I
Purification of AcsA

 
Substrate Specificity and Kinetic Properties—The Km and Vmax values for various acids were determined to examine the substrate specificity of AcsA (Table II). According to the catalytic efficiencies (kcat/Km), the best substrate for AcsA was isobutyric acid. The enzyme acted on butyric acid (C4), valeric acid (C5), and propionic acid (C3) with good catalytic rates but lower affinity. Formic acid (C1) and acetic acid (C2), or medium-chain fatty acids such as hexanoic acid (C6) and heptanoic acid (C7), were turned over more slowly and bound with much lower affinity by the enzyme. Aromatic acids (e.g. benzoic acid, 2-pyridinecarboxylic acid (picolinic acid), 3-pyridinecarboxylic acid (nicotinic acid), and 4-pyridinecarboxylic acid (isonicotinic acid)) and arylalkyl acids (e.g. 3-phenylpropionic acid and 4-phenylbutyric acid) were poor substrates for AcsA. Due to its substrate specificity, this enzyme can be termed a short-chain acyl-CoA synthetase. From linear Lineweaver-Burk plots, the apparent Km value for ATP was found to be 0.22 ± 0.02 mM, and for CoA a value of 0.37 ± 0.02 mM was calculated.


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  		TABLE II
Substrate specificity of AcsA

The reaction was carried out by the methods described under "Experimental Procedures." Apparent Km and apparent Vmax values were obtained from Lineweaver-Burk plots. NA, no activity could be detected.

 
Effects of Temperature and pH on the Activity and Stability of the Enzyme—The effects of pH and temperature on enzyme activity were examined. AcsA exhibited maximum activity at pH 7.5, as shown in Fig. 3A. The optimal reaction temperature appeared to be 35 °C when the reaction was carried out for 4 min (Fig. 3B).



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  		FIG. 3.
Effects of pH and temperature on the activity of AcsA. A, the reactions were carried out for 4 min at 35 °C in the following buffers (0.1 M): citrate/sodium citrate (•), potassium phosphate ({diamondsuit}), Tris/HCl ({blacksquare}), and NH4Cl/NH4OH ({blacktriangleup}). B, the reactions were carried out for 4 min at various temperatures. Relative activity is expressed as a percentage of the maximum activity attained under the experimental conditions used.

 
In contrast to determination of the temperature optimum of the reaction, which was performed for 4 min at different temperatures, protein stability was investigated after incubation for 30 min at various temperatures in 10 mM KP buffer (pH 7.5). An aliquot of the enzyme solution was taken, and then the enzyme activity was assayed under the standard conditions. It exhibited the following activity: 10 °C, 100%; 15 °C, 96%; 20 °C, 96%; 25 °C, 95%; 30 °C, 85%; 35 °C, 5.9%; and 40 °C, 0%.

The stability of AcsA was examined at various pH values. After the enzyme had been incubated at 25 °C for 30 min in the following buffers at a concentration of 0.32 M (citrate-NaOH buffer (pH 4.0–6.0), PIPES-NaOH buffer (pH 6.0–7.5), Tris/HCl buffer (pH 7.5–9.0), and glycine-NaOH buffer (pH 9.0–11.0)), an aliquot of the enzyme solution was taken, and then the enzyme activity was assayed under the standard conditions. AcsA was most stable in the pH range of 6.5–9.0, with 40% of its initial activity being retained even at pH 9.5.

Inhibitors of Enzyme Activity—Various compounds were investigated with regard to their inhibitory effects on enzyme activity (Table III). AcsA was very sensitive to AgNO3, CuCl, CuCl2, ZnCl2, CdCl2, HgCl2, NiCl2, and CoCl2. The addition of hydrophobic thiol-specific reagents such as p-chloromercuribenzoate, 5,5'-dithio-bis-2-nitrobenzoate, and N-ethylmaleimide caused strong inhibition of the activity of AcsA. These results suggest that, as reported for phenylacetyl-CoA ligase from Pseudomonas putida (47) and acyl-CoA synthetase from Penicillium chrysogenum (48), some SH groups of AcsA are essential for catalysis. By comparison, the hydrophilic thiolspecific reagent iodoacetate caused partial inhibition. The SH groups of AcsA involved in catalysis may be located in a hydrophobic environment, being inaccessible to hydrophilic iodoacetate. Carbonyl-specific reagents hardly inhibited the enzyme activity. AcsA was not sensitive to chelating agents such as {alpha},{alpha}'-dipyridyl, o-phenanthroline, 8-hydroxyquinoline, EDTA, and KCN, although diethyldithiocarbamate caused partial inhibition. Serine-modifying reagents, i.e. phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate, did not affect the activity at all.


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  		TABLE III
Effects of various compounds on the activity of AcsA

Each compound was added to the standard reaction mixture without the substrate, and then assaying of the enzyme was performed after adding the substrate. The final concentrations of the tested compounds were 1 mM, unless otherwise stated.

 
Complementation Analyses of an E. coli Acetyl-CoA Synthetase-deficient Mutant or Long-chain Acyl-CoA Synthetase-deficient Mutant—Another expression plasmid (pUC-acsA) was constructed and introduced into E. coli AJW1781 or K27. When the E. coli wild-type cells or the cells deficient in Acs (acetyl-CoA synthetase) (strain AJW1781) were inoculated into TB medium (37) and aerated at 37 °C, the wild-type cells and Acs-deficient cells grew at similar rates and excreted similar amounts of acetate before the mid-exponential phase. Although both types of cells continued to grow at similar rates, the extracellular acetate concentration differed. Whereas the concentration of extracellular acetate in the wild-type cells decreased to a minimum of <0.1 mM, that in the Acs-deficient cells did not decrease at all (37). We transformed AJW1781 with plasmid pUC-acsA, which causes the expression of AcsA. We cultured cells of the AJW1781 and transformant strains at 37 °C in TB medium, monitored their growth rates, and measured the extracellular acetate concentrations, respectively. The growth rate and behavior with respect to time course of the extracellular acetate concentration in AJW1781 harboring pUC-acsA were very similar to those in the AJW1781 strain (data not shown), demonstrating that AcsA was not able to complement E. coli acetyl-CoA synthetase (Acs). Next, we examined whether or not AcsA complements the FadD (long-chain acyl CoA synthetase)-deficient strain (K27). In contrast to the wild-type strain that grew in the medium containing a long-chain fatty acid (oleate) as the sole carbon and energy source, the K27 strain did not grow (38, 43). We transformed the K27 strain with pUC-acsA and plated the transformants on glucose or oleate minimal agar plates, respectively. The K27 cells harboring pUC-acsA were able to grow on glucose but not on oleate, demonstrating that AcsA did not complement E. coli long-chain acyl-CoA synthetase (FadD). Considering the substrate specificity of AcsA, it is reasonable that AcsA complements neither the E. coli acetyl-CoA synthetase-deficient mutant nor the long-chain acyl-CoA synthetase-deficient mutant.

Acyl-CoA Synthetase Activity in P. chlororaphis B23—To confirm the function of AcsA in vivo, P. chlororaphis B23 was grown in several culture media. When P. chlororaphis B23 was grown in medium containing 1% sucrose and 0.5% (NH4)2SO4 as the sole carbon and nitrogen sources, respectively, AcsA activity was not observed. These cells also lacked aldoxime dehydratase and amidase activities and exhibited only 0.142 unit/mg NHase activity. However, on replacement of sucrose and (NH4)2SO4 with butyraldoxime as the sole carbon and nitrogen source, which was previously reported to be an inducer of aldoxime dehydratase (33), not only aldoxime dehydratase but also NHase, amidase, and AcsA were induced. The obtained specific activity values were as follows: aldoxime dehydratase, 0.878 unit/mg; NHase, 66.2 unit/mg; amidase, 0.0749 unit/mg; and AcsA, 1.12 units/mg. To identify the expression of all four enzymes, cell-free extracts (10 µg) of P. chlororaphis B23 grown with butyraldoxime were electroblotted from the SDS-PAGE gels onto a polyvinylidene difluoride membrane. The amino acid sequences of the protein bands indicated (Fig. 4, arrows) were then analyzed. The NH2-terminal amino acid sequences of each band indicated (Fig. 4, arrows) were the same as those of AcsA (MRDYEHVVE), amidase (AITRPTLD), aldoxime dehydratase (MESAIDTHL), and {alpha} and {beta} subunits of NHase (mixture of STSISTTA and MDGFHDLG), respectively. These findings indicate that AcsA had to be inducibly co-translated with other enzymes to utilize butyraldoxime as a carbon and nitrogen source and/or to generate energy. On the other hand, neither NhpC nor NhpS activity was measured because the role of each remains unknown, and neither gene product was purified in this study. Thus, it is unclear whether or not the formation of NhpC and NhpS is also co-regulated.



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  		FIG. 4.
SDS-PAGE of cell-free extracts of P. chlororaphis B23. SDS-PAGE was performed in a 10% polyacrylamide slab gel. Protein bands were detected by staining with Coomassie Brilliant Blue. The arrows indicate the bands corresponding to AcsA, amidase, aldoxime dehydratase, and NHase, respectively. Lane M, marker proteins; lane 1, cell-free extracts (10 µg) of P. chlororaphis B23 grown without aldoxime; lane 2, cell-free extracts (10 µg) of P. chlororaphis B23 grown with butyraldoxime (0.05%) in place of sucrose and (NH4)2SO4. The composition of each medium is given under "Experimental Procedures."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The biological metabolism of nitriles in various living organisms such as microorganisms, plants, insects, and animals has been studied for a long time. In both the nitrilase pathway and the NHase/amidase pathway, acid and ammonium are produced as enzymatic reaction products. However, there has been no report on further metabolism of an acid produced from the corresponding nitrile. Sequence analysis of the downstream region of the amidase and NHase genes in the "industrial microorganism" P. chlororaphis B23 revealed the existence of an ORF (acsA) exhibiting sequence similarity to the acyl-CoA synthetase gene, whose protein ligates an acid and CoA to synthesize acyl-CoA. Expression of acsA in E. coli and purification and characterization of AcsA revealed that AcsA is an acyl-CoA synthetase. Our work is the first demonstration of a gene cluster consisting of the structural genes of aldoxime dehydratase, amidase, NHase, and acyl-CoA synthetase involved in the consecutive metabolism of nitriles.

Early in the course of the purification of AcsA, we found that loss of AcsA activity occurred on dialysis and that reversible restoration of the enzyme activity followed the addition of salt to the assay mixture. The salt activation of another acyl-CoA synthetase from E. coli was described previously (49). The reaction rate of this E. coli enzyme depends on the salt concentration, with 2.5-fold enhancement of the enzyme activity being observed on the addition of 100 mM (NH4)2SO4. On the contrary, AcsA exhibited much higher activity (>12-fold; an increase from 7.9% to 99%) with the addition of 100 mM (NH4)2SO4 in comparison with activity without the addition of any (NH4)2SO4. These findings suggest that AcsA is more subject to the salt effect on the solution structure of the water surrounding the enzyme than the acyl-CoA synthetase from E. coli.

From Pseudomonas aeruginosa, which belongs to the same genus as P. chlororaphis B23, butyryl-CoA synthetase has been purified and characterized in detail (50). AcsA is similar to this butyryl-CoA synthetase in the following characteristics. Both enzymes act well on acids with carbon chain lengths of 3–5, but not on acids with carbon chain lengths of >6. Moreover, both enzymes are inducibly formed in the respective microbial cells when the microorganisms are grown with butyric acid as the major carbon source. On the other hand, there are some differences between the two enzymes. The butyryl-CoA synthetase has a molecular mass of ~142 kDa and consists of four identical subunits (one of which has a molecular mass of about 37 kDa). On the contrary, the calculated molecular mass of 60,210 for the AcsA monomer was verified by MALDI-TOF mass spectrometry, suggesting that it is different from the butyryl-CoA synthetase subunit in molecular mass. Moreover, the Vmax (15.1 ± 0.8 units/mg) of AcsA toward the same substrate, butyric acid, is considerably higher than that of butyryl-CoA synthetase (1.03 units/mg). It will be interesting to perform detailed comparative studies on the two enzymes, particularly with respect to molecular mass and kinetic rate.

AcsA of P. chlororaphis B23 showed significant activity toward butyric acid, with butyryl-CoA being yielded. In the case of the aldoxime dehydratase (OxdA) of this strain (33), the kcat value for butyraldoxime (5.4 min–1) is much higher than that for pyridine-4-aldoxime (0.090 min–1), and OxdA prefers aliphatic aldoximes to aromatic ones as substrates among the tested aldoximes. For the NHase of this strain (29), butyronitrile is a better substrate and exhibits the highest affinity (Km = 1.03 mM) among all of the tested nitriles. Other aliphatic nitriles such as propionitrile and acrylonitrile are remarkably active as substrates for the enzyme, although aromatic nitriles are barely hydrated by the NHase (29). The amidase linked with this NHase can also act on aliphatic amides including butyramide and isobutyramide, rather than aromatic amides, as substrates (51). In this study, the kinetic parameters of AcsA indicate that aliphatic acids are more effective substrates than aromatic ones. Here, we observed a similar trend in substrate specificity among the OxdA, NHase, amidase, and AcsA, the genes of which coexist with the same orientation in the same gene cluster in P. chlororaphis B23 (Fig. 1). To confirm the function of AcsA in vivo, we measured the activity of each of aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase in cell-free extracts of P. chlororaphis B23 grown in several culture media. When this strain was grown in medium containing 1% sucrose and 0.5% (NH4)2SO4 as the sole carbon and nitrogen sources, respectively, the four enzyme activities were negligible or absent. When butyraldoxime (0.05%) was used as the sole carbon and nitrogen source, the activities of all four enzymes toward the corresponding substrates derived from butyraldoxime (i.e. butyraldoxime, butyronitrile, butyramide, and butyric acid) were detected, and the expression of all four enzymes was confirmed by NH2-terminal amino acid sequencing analyses for each induced protein band in SDS-PAGE (Fig. 4). These findings indicated that these four enzymes are sequentially correlated with one another in vivo (Fig. 4). On the other hand, this strain did not grow when oleate (1%) was used as the carbon source, suggesting that an enzyme capable of esterifying long-chain fatty acids was not expressed in this bacterium. Furthermore, we detected significant acetyl-CoA synthetase activity (0.211 unit/mg) but slight butyryl-CoA synthetase activity (0.025 unit/mg) when acetate was used as the carbon source, suggesting that an acetyl-CoA synthetase activity that was not due to AcsA was detected in this bacterium for growth on acetate. These findings also support that the formation of AcsA is essential for the further utilization of an acid produced from an aldoxime, although P. chlororaphis B23 has at least two enzymes capable of esterifying fatty acids, namely, short-chain acyl-CoA synthetase (AcsA) and acetyl-CoA synthetase. We demonstrate here for the first time an overall nitrile pathway (aldoxime->nitrile->amide->acid->acyl-CoA) comprising the above-mentioned four enzymes. The acyl-CoA would be further used as carbon and energy sources via the {beta}-oxidation pathway. In order to metabolize the acids derived through the nitrile pathway, AcsA is vital for nitrile-degrading microorganisms. During the course of evolution, P. chlororaphis B23 might have acquired this gene arrangement for efficient nitrile degradation. Here, we have a question: what is the natural source of aldoxime that should be the natural substrate for the first enzyme in the nitrile pathway? In glucosinolate-producing plants (e.g. Arabidopsis), aldoximes are formed from amino acids by cytochrome P450 belonging to the CYP79 family (52–55). We are interested in the biosynthesis of a substrate (of aldoxime dehydratase), which remains undetermined. Further sequence analyses of the upstream region of the nitrile pathway gene cluster in P. chlororaphis B23 may provide information on such aldoxime biosynthesis.

Further downstream of nhpC, we sequenced the complete ORF, nhpS. To date, we have found neither an ORF that shows amino acid sequence similarity to NhpS nor an acyl-CoA synthetase gene in various types of gene organization involved in nitrile metabolism by nitrile-degrading microorganisms other than P. chlororaphis B23. The overall gene organization involved in nitrile metabolism by P. chlororaphis B23 is different from those in other nitrile-degrading microorganisms, and there has been no report of a gene cluster that is identical or very similar to that of P. chlororaphis B23. To our knowledge, this is also the first report that the enzyme genes involved in "catabolism" resulting in acid generation and "anabolism" utilizing acids coexist in the same gene cluster. Although the function of NhpS is unclear, the existence of nhpS in this cluster makes us speculate that this protein may be involved in the nitrile pathway. To clarify the role of NhpS, additional studies are required.

Recently, special attention has been paid to long-chain acyl-CoA serving as an important molecule. In yeast, long-chain acyl-CoA has been reported to affect not only lipid biosynthesis and fatty acid degradation but also cellular systems and functions, including ion channels, ion pumps, translocators, enzymes, membrane fusion, and gene regulation (56). In E. coli, long-chain acyl-CoA, but not short-chain acyl-CoA or fatty acids, acts as a regulatory molecule in the derepression of the genes encoding proteins required for the transport and degradation of medium- and long-fatty acids. On the other hand, these genes are also positively controlled by cAMP and cAMP receptor protein (57, 58). Contrary to the above phenomena in E. coli, the expression of the acsA gene is positively controlled by an aldoxime or amide in P. chlororaphis B23. Moreover, the finding that the induction of AcsA (0.873 unit/mg) is observed when this strain is cultured in the presence of butyric acid indicates that acsA expression is also controlled by butyric acid or a derivative of it produced enzymatically (e.g. butyryl-CoA). Thus, remarkable differences in the regulation mechanisms for each acyl-CoA synthetase gene are observed between E. coli and P. chlororaphis B23. To date, the role of short-chain acyl-CoA as a regulatory molecule has not been demonstrated to the best of our knowledge. The regulation mechanism for the gene cluster including the structural genes of the aldoxime dehydratase, NHase, amidase, and acyl-CoA synthetase of P. chlororaphis B23 is an interesting target because a variety of molecules, namely, aldoximes, amides, and acids (or acyl-CoA), involved in the nitrile pathway act as inducers for all four enzymes. Thus, further analyses, including the isolation of a regulatory protein that plays a key role in control of the expression of this gene cluster, are in progress in order to obtain a better overview of the nitrile pathway.


   FOOTNOTES
 
* This work was supported in part by the 21st Century COE Program (www.tara.tsukuba.ac.jp/~coe21/) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan; a grant-in-aid for scientific research from MEXT; the Industrial Technology Research Grant Program in 2002 of the New Energy and Industrial Technology Development Organization (NEDO) of Japan; the National Project on Protein Structural and Functional Analyses; and Research Grant (A) of the University Research Projects. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" 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 DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB125061 [GenBank] . Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed. Fax: 81-29-853-4605 (Institute).

1 The abbreviations used are: NHase, nitrile hydratase; ORF, open reading frame; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PIPES, 1,4-piperazinediethanesulfonic acid; AcsA, acyl-CoA synthetase. Back


   ACKNOWLEDGMENTS
 
We thank Hiroshi Fukatsu (The University of Tsukuba) for kind discussion.



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