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Molecular and enzymatic analysis of the “aldoxime–nitrile pathway” in the glutaronitrile degrader Pseudomonas sp. K-9

  • Applied Microbial and Cell Physiology
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Abstract

A gene cluster responsible for aldoxime metabolism in the glutaronitrile degrader Pseudomonas sp. K-9 was analyzed genetically and enzymatically. The cluster was composed of genes coding for aldoxime dehydratase (Oxd), nitrile hydratase (NHase), NHase activator, amidase, acyl-CoA ligase, and some regulatory and functionally unknown proteins, which were similar to proteins appearing in the “aldoxime–nitrile pathway” gene cluster from strains having Fe-containing NHase. A key enzyme in the cluster, OxdK, which has 32.7–90.3 % identity with known Oxds, was overexpressed in Escherichia coli cells under the control of a T7 promoter in its His6-tagged form, purified, and characterized. The enzyme showed similar characteristics with the known Oxds coexisting with an Fe-containing NHase in its subunit structure, substrate specificity, and effects on various compounds. The enzyme can be classified into a group of “aliphatic aldoxime dehydratase (EC 4.99.1.5).” The existence of a gene cluster of enzymes responsible for aldoxime metabolism via the aldoxime–nitrile pathway (aldoxime→nitrile→amide→acid→acyl-CoA) in Pseudomonas sp. K-9, and the fact that the proteins comprising the cluster are similar to those acting on aliphatic type substrates, evidently clarified the alkylaldoxime-degrading pathway in that strain.

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References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Asano Y (2002) Overview of screening for new microbial catalysts and their uses in organic synthesis—selection and optimization of biocatalysts. J Biotechnol 94:65–72

    Article  CAS  Google Scholar 

  • Asano Y, Kato Y (1998) Z-Phenylacetaldoxime degradation by a novel aldoxime dehydratase from Bacillus sp. strain OxB-1. FEMS Microbiol Lett 158:185–190

    Article  CAS  Google Scholar 

  • Asano Y, Tani Y, Yamada H (1980) A new enzyme “nitrile hydratase” which degrades acetonitrile in combination with amidase. Agric Biol Chem 44:2251–2252

    CAS  Google Scholar 

  • Asano Y, Fujishiro K, Tani Y, Yamada H (1982a) Aliphatic nitrile hydratase from Arthrobacter sp. J-1. Purification and characterization. Agric Biol Chem 46:1165–1174

    CAS  Google Scholar 

  • Asano Y, Yasuda T, Tani Y, Yamada H (1982b) A new enzymatic method of acrylamide production. Agric Biol Chem 46:1183–1189

    CAS  Google Scholar 

  • Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44

    Article  CAS  Google Scholar 

  • Barbe V, Vallenet D, Fonknechten N, Kreimeyer A, Oztas S, Labarre L, Cruveiller S, Robert C, Duprat S, Wincker P, Ornston LN, Weissenbach J, Marliere P, Cohen GN, Medigue C (2004) Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res 32:5766–5779

    Article  CAS  Google Scholar 

  • Black PN, DiRusso CC (2003) Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification. Microbiol Mol Biol Rev 67:454–472

    Article  CAS  Google Scholar 

  • Cowan DA, Cameron RA, Tsekoa TL (2003) Comparative biology of mesophilic and thermophilic nitrile hydratases. Adv Appl Microbiol 52:123–158

    Article  CAS  Google Scholar 

  • Davis BJ (1964) Disc electrophoresis II. Methods and application to human serum proteins. Ann N Y Acad Sci 121:404–427

    Article  CAS  Google Scholar 

  • Hann EC, Eisenberg A, Fager SK, Perkins NE, Gallagher FG, Cooper SM, Gavagan JE, Stieglitz B, Hennessey SM, DiCosimo R (1999) 5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23. Bioorg Med Chem 7:2239–2245

    Article  CAS  Google Scholar 

  • Hashimoto Y, Hosaka H, Oinuma KI, Goda M, Higashibata H, Kobayashi M (2005) Nitrile pathway involving Acyl-CoA synthetase: overall metabolic gene organization, and purification and characterization of the enzyme. J Biol Chem 280:8660–8667

    Article  CAS  Google Scholar 

  • Kato Y, Asano Y (2003) High-level expression of a novel FMN-dependent heme-containing lyase, phenylacetaldoxime dehydratase of Bacillus sp. strain OxB-1, in heterologous hosts. Protein Expr Purif 28:131–139

    Article  CAS  Google Scholar 

  • Kato Y, Ooi R, Asano Y (1998) Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes. Arch Microbiol 170:85–90

    Article  CAS  Google Scholar 

  • Kato Y, Tsuda T, Asano Y (1999a) Nitrile hydratase involved in aldoxime metabolism from Rhodococcus sp. strain YH3-3 purification and characterization. Eur J Biochem 263:662–670

    Article  CAS  Google Scholar 

  • Kato Y, Ooi R, Asano Y (1999b) A new enzymatic method of nitrile synthesis by Rhodococcus sp. strain YH3-3. J Mol Catal, B Enzym 6:249–256

    Article  CAS  Google Scholar 

  • Kato Y, Ooi R, Asano Y (2000a) Distribution of aldoxime dehydratase in microorganisms. Appl Environ Microbiol 66:2290–2296

    Article  CAS  Google Scholar 

  • Kato Y, Nakamura K, Sakiyama H, Mayhew SG, Asano Y (2000b) A novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of the gene. Biochemistry 39:800–809

    Article  CAS  Google Scholar 

  • Kato Y, Yoshida S, Xie S-X, Asano Y (2004) Aldoxime dehydratase co-existing with nitrile hydratase and amidase in iron-type nitrile hydratase producer Rhodococcus sp. N-771. J Biosci Bioeng 97:250–259

    Article  CAS  Google Scholar 

  • Kato Y, Yoshida S, Asano Y (2005) Polymerase chain reaction for identification of aldoxime dehydratase in aldoxime- or nitrile-degrading microorganisms. FEMS Microbiol Lett 246:243–249

    Article  CAS  Google Scholar 

  • Kobayashi K, Yoshioka S, Kato Y, Asano Y, Aono S (2005) Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex. J Biol Chem 280:5486–5490

    Article  CAS  Google Scholar 

  • Konishi K, Ishida K, Oinuma K, Ohta T, Hashimoto Y, Higashibata H, Kitagawa T, Kobayashi M (2004) Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase. J Biol Chem 279:47619–47625

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Nishiyama M, Horinouchi S, Kobayashi M, Nagasawa T, Yamada H, Beppu T (1991) Cloning and characterization of genes responsible for metabolism of nitrile compounds from Pseudomonas chlororaphis B23. J Bacteriol 173:2465–2472

    Article  CAS  Google Scholar 

  • Oinuma K, Hashimoto Y, Konishi K, Goda M, Noguchi T, Higashibata H, Kobayashi M (2003) Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23. Sequencing, gene expression, purification, and characterization. J Biol Chem 278:29600–29608

    Article  CAS  Google Scholar 

  • Oinuma KI, Ohta T, Konishi K, Hashimoto Y, Higashibata H, Kitagawa T, Kobayashi M (2004) Heme environment in aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis. FEBS Lett 568:44–48

    Article  CAS  Google Scholar 

  • Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A 85:2444–2448

    Article  CAS  Google Scholar 

  • Qing G, Ma L-C, Khorchid A, Swapna GVT, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22:877–882

    Article  CAS  Google Scholar 

  • Ropp JD, Gunsalus IC, Sligar SG (1993) Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita. J Bacteriol 175:6028–6037

    Article  CAS  Google Scholar 

  • Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619–629

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA

    Google Scholar 

  • Sibbessen Ö, Koch B, Rouzé P, Mϕller BL, Halkier BA (1995) Biosynthesis of cyanogenic glucosides. Elucidation of the pathway and characterization of the cytochromes P-450involved. In: Wallsgrove RM (ed) Amino acids and their derivatives in higher plants. Society for experimental biology seminar series, vol 56. Cambridge University Press, Cambdridge, pp 227–241

    Chapter  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  Google Scholar 

  • Toyama H, Fujii T, Aoki N, Matsushita K, Adachi O (2003) Molecular cloning of quinohemoprotein alcohol dehydrogenase, ADH IIB, from Pseudomonas putida HK5. Biosci Biotechnol Biochem 67:1397–1400

    Article  CAS  Google Scholar 

  • Xie S-X, Kato Y, Asano Y (2001) High yield synthesis of nitriles by a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Biosci Biotechnol Biochem 65:2666–2672

    Article  CAS  Google Scholar 

  • Xie S-X, Kato Y, Komeda H, Yoshida S, Asano Y (2003) A novel gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4. Biochemistry 42:12056–12066

    Article  CAS  Google Scholar 

  • Yamada H, Kobayashi M (1996) Nitrile hydratase and its application to industrial production of acrylamide. Biosci Biotechnol Biochem 60:1391–1400

    Article  CAS  Google Scholar 

  • Yamada H, Asano Y, Tani Y (1980) Microbial utilization of glutaronitrile. J Ferment Technol 58:495–500

    CAS  Google Scholar 

Download references

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  1. Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama, 939-0398, Japan

    Yasuo Kato & Yasuhisa Asano

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  1. Yasuo Kato
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  2. Yasuhisa Asano
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Correspondence to Yasuhisa Asano.

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Kato, Y., Asano, Y. Molecular and enzymatic analysis of the “aldoxime–nitrile pathway” in the glutaronitrile degrader Pseudomonas sp. K-9. Appl Microbiol Biotechnol 70, 92–101 (2006). https://doi.org/10.1007/s00253-005-0044-4

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  • DOI: https://doi.org/10.1007/s00253-005-0044-4

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