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Aldoxime Dehydratase Co-Existing with Nitrile Hydratase and Amidase in the Iron-Type Nitrile Hydratase- Producer Rhodococcus Sp

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Aldoxime Dehydratase Co-Existing with Nitrile Hydratase and Amidase in the Iron-Type Nitrile Hydratase- Producer Rhodococcus Sp JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 97, No. 4, 250–259. 2004 Aldoxime Dehydratase Co-existing with Nitrile Hydratase and Amidase in the Iron-Type Nitrile Hydratase- Producer Rhodococcus sp. N-771 YASUO KATO,1 SATOSHI YOSHIDA,1 SHENG-XUE XIE,1 AND YASUHISA ASANO1* Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan1 Received 9 October 2003/Accepted 26 January 2004 We identified an aldoxime dehydratase (Oxd) gene in the 5>-flanking region of the nitrile hy- dratase–amidase gene cluster in the photoreactive iron-type nitrile hydratase-producer, Rhodo- coccus sp. N-771. The enzyme showed 96.3%, 77.6%, and 30.4% identities with the Oxds of Rhodococcus globerulus A-4, Pseudomonas chlororaphis B23, and Bacillus sp. OxB-1, respectively. The enzyme was expressed in Escherichia coli under the control of the lac- or T7 promoters in its intact and His6-tagged forms, purified, and characterized. The enzyme had heme b as a prosthetic group, catalyzed a stoichiometric dehydration of aldoxime into nitrile, and exhibited the highest activity at neutral pH and at around 30LC similar to the known Oxd from Bacillus sp. OxB-1. The activity was enhanced by reducing agents, such as Na2S, Na2S2O4, 2-mercaptoethanol, and L-cys- teine and supplementary additions of electron acceptors such as flavins, sulfite ion, and vitamin K3. The effect of various chemicals on the enzyme activity was different in the presence and ab- sence of the reducing reagent, Na2S. The enzyme preferentially acts on aliphatic-type substrates and the substrate specificity of the enzyme coincides with that reported for nitrile hydratase pro- duced by the strain. [Key words: aldoxime dehydratase, nitrile hydratase, amidase, aldoxime–nitrile pathway, Rhodococcus] In nature, two distinct pathways for the degradation of by a combination of a novel aldoxime dehydratase (Oxd; nitriles to carboxylic acids exist (1, 2). Nitrilase (Nit) cata- EC 4.2.1.–) and nitrile-degrading enzymes such as NHase lyzes the direct hydrolysis of nitrile to carboxylic acid (3), and Nit (Fig. 1) (2, 12–14). It has been shown that Oxd, while nitrile hydratase (NHase) catalyzes the hydration of which is widely distributed in microorganisms, co-exists nitrile to amide (1–6), which is then hydrolyzed to carbox- with the nitrile-degrading enzymes and functions to metab- ylic acid by amidase (7). NHase in particular has been wide- olize aldoximes (14, 15). The Oxds are used for the novel ly studied because it is used as a biocatalyst in various enzymatic syntheses of nitrile from aldoxime under mild chemical industries; i.e., the industrial production of acryl- conditions (13, 16, 17). We purified and characterized Oxd amide, nicotinamide, and 5-cyanovaleramide (1–4, 8–11). from Bacillus sp. strain OxB-1, cloned the gene (oxd) and Despite the importance of the enzyme for industrial use, in- overexpressed that in Escherichia coli, and showed that the formation about its biological functions is quite limited. oxd gene is genetically linked with a Nit gene (nit) in the ge- Recently, we have been successful in clarifying the mi- nome of the strain (18, 19). On the other hand, however, the crobial metabolism of aldoximes. We isolated aldoxime-de- possible enzymological and genetic relationships between grading microorganisms and confirmed the metabolism of Oxd and NHase still remain unclear. We have purified Oxd aldoximes via nitriles into the corresponding carboxylic acids from Rhodococcus globerulus A-4 that degrades aldoximes into carboxylic acids via Oxd, NHase, and amidase and the * Corresponding author. e-mail: [email protected] gene of the enzyme was cloned and sequenced (20). We phone: +81-(0)766-56-7500 fax: +81-(0)766-56-2498 found that the enzyme has 94% identity with a short poly- Abbreviations: C-His6-tagged OxdRE, His6-tagged aldoxime de- peptide (137 amino acids), which had not been assigned as hydratase from Rhodococcus sp. N-771 at its C-terminus; DMSO, di- a protein, coded by the 5>-flanking region of the NHase and methyl sulfoxide; IPTG, isopropyl--D-thiogalactopyranoside; NHase, amidase gene cluster of Rhodococcus sp. N-771: the strain nitrile hydratase; N-His6-tagged OxdRE, His6-tagged aldoxime dehy- dratase from Rhodococcus sp. N-771 at its N-terminus; Nit, nitrilase; had been isolated based on its ability to degrade acrylo- ORF, open reading frame; OxdA, aldoxime dehydratase from Pseu- nitrile and as a source of an iron-type NHase (21, 22; domonas chlororaphis B23; OxdB, phenylacetaldoxime dehydratase Watanabe, I., Sato, S., and Takano, T., Japan patent S56- from Bacillus sp. OxB-1; OxdRE, aldoxime dehydratase from Rhodo- coccus sp. N-771; OxdRG, alkylaldoxime dehydratase from Rhodo- 17918). Very recently, an Oxd gene (oxdA) was cloned from coccus globerulus A-4; PAOx, phenylacetaldoxime; PyOx, pyridine-3- Pseudomonas chlororaphis B23, which had been isolated as aldoxime. an NHase-producer by one of the authors (9), sequenced, 250 VOL. 97, 2004 ALDOXIME DEHYDRATASE FROM NITRILE HYDRATASE-PRODUCER 251 FIG. 1. Aldoxime–nitrile pathway in microorganisms. and its gene product (OxdA) characterized (23). We are in- yeast extract, and 1% NaCl, pH 7.5) or MMI medium (1.25% terested in comparing the properties of Oxds from various Bacto Tryptone, 2.5% Bacto yeast extract, 0.85% NaCl, 20 mM origins to understand the structure and function of Oxds. Tris–HCl [pH 7.5] and 0.4% glycerol) containing the appropriate In this report, we cloned and sequenced a gene coding amount of antibiotics. For the induction of the gene under the con- D Oxd located in the 5>-flanking region of the NHase and ami- trol of lac- or T7 promoters, 1 mM of isopropyl - -thiogalacto- pyranoside (IPTG) was added to the LB medium. dase gene cluster in Rhodococcus sp. N-771. The enzyme Enzyme assay The Oxd activity was assayed by measuring was expressed in E. coli, purified, characterized, and its the formation of nitriles from the corresponding aldoximes. The properties were compared with those of known Oxds. We standard assay solution contained 50 mM potassium phosphate report here the existence and properties of an Oxd geneti- buffer (KPB, pH 7.0), 5 mM aldoxime, 10% (v/v) DMSO, 1 mM cally linked to NHase and amidase in a strain isolated as a Na2S, and the enzyme solution in a final volume of 0.5 ml. After nitrile-degrader. the reaction was run at 30LC for 10 min during which the reaction proceeded linearly, 0.5 ml of 0.5 M H3PO4 was added to stop the reaction. The supernatant obtained by centrifugation (15,000Pg, MATERIALS AND METHODS 10 min) was assayed by HPLC using an ODS-80Ts column and a mobile phase of aqueous CH3CN containing 10 mM H3PO4 deliv- Materials DEAE- and Butyl-Toyopearl, and the HPLC ered at a flow rate of 1.0 ml/min and monitored at 254 nm or by column ODS-80Ts were purchased from Tosoh (Tokyo). Stan- gas-liquid chromatography (GLC; Shimadzu, Kyoto) equipped dard proteins for the native- and SDS–PAGE were obtained from with a flame ionization detector with a glass column (2 mm by Dai-ichi Chem. (Tokyo) and Amersham Biosciences (Uppsala, 2 m) packed with polyethylene glycol (PEG20M, 60–80 mesh; Sweden), respectively. The aldoximes were prepared from their GL-Science, Tokyo). One unit (U) of the enzyme activity is de- corresponding aldehydes and hydroxylamine according to a pre- fined as the amount of enzyme that catalyzes the conversion of the viously described method (12, 13, 17). Restriction enzymes and substrate to the product at a rate of 1 mol/min. Mean values from DNA-modifying enzymes were purchased from Takara (Tokyo), two experiments are shown in the text. Toyobo (Osaka), New England Biolabs. (Beverly, MA, USA), General recombinant DNA techniques The plasmid DNA Roche (Mannheim, Germany), and MBI Fermentas (Vilnius, was isolated by a PI-100 Automatic Plasmid Isolation System Lithuania) and used according to the manufacturers’ protocols. (Kurabo, Osaka) or by a plasmid purification kit from Qiagen Bacto Tryptone and Bacto yeast extract were from Difco (Detroit, (Valencia, CA, USA). The other general procedures were per- MI, USA). All other chemicals were from commercial sources and formed as described by Sambrook et al. (26). The nucleotide se- used without further purification. quence was determined by the dideoxy-chain termination method Analytical methods The native- and SDS–PAGE were car- (27) using a SequiTherm EXCEL™II Long-Read™ DNA Sequenc- ried out as described by Davis (24) and Laemmli (25), respec- ing Kit LC (Epicenter Technologies, Madison, WI, USA) or Thermo tively, with an electrophoresis model unit made by ATTO (Tokyo). Sequenase Cycle Sequencing Kit (Amersham, Cleveland, OH, The molecular weight (Mr) of the enzyme was determined as previ- USA) and a 4000L DNA autosequencer (LI-COR, Lincoln, NB, ously described (18). USA). A homology search was performed using the sequence Bacterial strains, plasmids and culture conditions Rhodo- similarity searching programs FASTA (28) and BLAST (29). The coccus sp. N-771 (FERM-P4445) was used as the source of the ClustalW method was used to align the sequences (30). The Pwo DNA throughout this study. We re-identified the strain and clas- polymerase-mediated PCR amplification was carried out in a re- sified it as Rhodococcus erythropolis based on an analysis of its action mixture that contained 25 ng of template DNA, 100 pmol of fatty acid composition and 16S rRNA sequence similarities. Major each primer, and 0.5 U of Pwo polymerase in a final volume of fatty acids present were tetradecanoic, pentadecanoic, anteiso-pen- 50 l. Thirty thermal cycles were employed, each consisting of tadecanoic, -7-cis-hexadecenoic, hexadecanoic, -8-cis-hepta- 95LC for 1 min, 50LC for 1.5 min, and 72LC for 2.5 min.
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