Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Nov;173(21):6694–6704. doi: 10.1128/jb.173.21.6694-6704.1991

Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase.

J F Mayaux 1, E Cerbelaud 1, F Soubrier 1, P Yeh 1, F Blanche 1, D Pétré 1
PMCID: PMC209017  PMID: 1938876

Abstract

A new enantiomer-selective amidase active on several 2-aryl propionamides was identified and purified from a newly isolated Rhodococcus strain. The characterized amidase is an apparent homodimer, each molecule of which has an Mr of 48,554; it has a specific activity of 16.5 mumol of S(+)-2-phenylpropionic acid formed per min per mg of enzyme from the racemic amide under our conditions. An oligonucleotide probe was deduced from limited peptide information and was used to clone the corresponding gene, named amdA. As expected, significant homologies were found between the amino acid sequences of the enantiomer-selective amidase of Rhodococcus sp., the corresponding enzyme from Brevibacterium sp. strain R312, and several known amidases, thus confirming the existence of a structural class of amidase enzymes. Genes probably coding for the two subunits of a nitrile hydratase, albeit in an inverse order, were found 39 bp downstream of amdA, suggesting that such a genetic organization might be conserved in different microorganisms. Although we failed to express an active Rhodococcus amidase in Escherichia coli, even in conditions allowing the expression of an active R312 enzyme, the high-level expression of the active recombinant enzyme could be demonstrated in Brevibacterium lactofermentum by using a pSR1-derived shuttle vector.

Full text

PDF
6704

Images in this article

6702FIG. 5
on p.6702

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  2. Brammar W. J., Charles I. G., Matfield M., Liu C. P., Drew R. E., Clarke P. H. The nucleotide sequence of the amiE gene of Pseudomonas aeruginosa. FEBS Lett. 1987 May 11;215(2):291–294. doi: 10.1016/0014-5793(87)80164-3. [DOI] [PubMed] [Google Scholar]
  3. Chang T. H., Abelson J. Identification of a putative amidase gene in yeast Saccharomyces cerevisiae. Nucleic Acids Res. 1990 Dec 11;18(23):7180–7180. doi: 10.1093/nar/18.23.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ciora T., Denèfle P., Mayaux J. F. Rapid one-step automated sequencing reactions for 16 DNA samples using Taq polymerase and fluorescent primers. Nucleic Acids Res. 1991 Jan 11;19(1):188–188. doi: 10.1093/nar/19.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Corrick C. M., Twomey A. P., Hynes M. J. The nucleotide sequence of the amdS gene of Aspergillus nidulans and the molecular characterization of 5' mutations. Gene. 1987;53(1):63–71. doi: 10.1016/0378-1119(87)90093-x. [DOI] [PubMed] [Google Scholar]
  6. Denèfle P., Kovarik S., Guitton J. D., Cartwright T., Mayaux J. F. Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form. Gene. 1987;56(1):61–70. doi: 10.1016/0378-1119(87)90158-2. [DOI] [PubMed] [Google Scholar]
  7. Hashimoto Y., Nishiyama M., Ikehata O., Horinouchi S., Beppu T. Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli. Biochim Biophys Acta. 1991 Feb 16;1088(2):225–233. doi: 10.1016/0167-4781(91)90058-t. [DOI] [PubMed] [Google Scholar]
  8. Ikehata O., Nishiyama M., Horinouchi S., Beppu T. Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli. Eur J Biochem. 1989 May 15;181(3):563–570. doi: 10.1111/j.1432-1033.1989.tb14761.x. [DOI] [PubMed] [Google Scholar]
  9. Jung G., Denèfle P., Becquart J., Mayaux J. F. High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1 beta. Ann Inst Pasteur Microbiol. 1988 Jan-Feb;139(1):129–146. doi: 10.1016/0769-2609(88)90100-7. [DOI] [PubMed] [Google Scholar]
  10. Latta M., Philit M., Maury I., Soubrier F., Denèfle P., Mayaux J. F. Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli. DNA Cell Biol. 1990 Mar;9(2):129–137. doi: 10.1089/dna.1990.9.129. [DOI] [PubMed] [Google Scholar]
  11. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  12. Mayaux J. F., Cerebelaud E., Soubrier F., Faucher D., Pétré D. Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase. J Bacteriol. 1990 Dec;172(12):6764–6773. doi: 10.1128/jb.172.12.6764-6773.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Parsot C., Cohen G. N. Cloning and nucleotide sequence of the Bacillus subtilis hom gene coding for homoserine dehydrogenase. Structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II. J Biol Chem. 1988 Oct 15;263(29):14654–14660. [PubMed] [Google Scholar]
  14. Parsot C. Evolution of biosynthetic pathways: a common ancestor for threonine synthase, threonine dehydratase and D-serine dehydratase. EMBO J. 1986 Nov;5(11):3013–3019. doi: 10.1002/j.1460-2075.1986.tb04600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Schröder G., Waffenschmidt S., Weiler E. W., Schröder J. The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem. 1984 Jan 16;138(2):387–391. doi: 10.1111/j.1432-1033.1984.tb07927.x. [DOI] [PubMed] [Google Scholar]
  16. Sekine M., Watanabe K., Syono K. Nucleotide sequence of a gene for indole-3-acetamide hydrolase from Bradyrhizobium japonicum. Nucleic Acids Res. 1989 Aug 11;17(15):6400–6400. doi: 10.1093/nar/17.15.6400. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Staden R., McLachlan A. D. Codon preference and its use in identifying protein coding regions in long DNA sequences. Nucleic Acids Res. 1982 Jan 11;10(1):141–156. doi: 10.1093/nar/10.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Staden R. Measurements of the effects that coding for a protein has on a DNA sequence and their use for finding genes. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):551–567. doi: 10.1093/nar/12.1part2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Staden R. Methods to define and locate patterns of motifs in sequences. Comput Appl Biosci. 1988 Mar;4(1):53–60. doi: 10.1093/bioinformatics/4.1.53. [DOI] [PubMed] [Google Scholar]
  20. Tsuchiya K., Fukuyama S., Kanzaki N., Kanagawa K., Negoro S., Okada H. High homology between 6-aminohexanoate-cyclic-dimer hydrolases of Flavobacterium and Pseudomonas strains. J Bacteriol. 1989 Jun;171(6):3187–3191. doi: 10.1128/jb.171.6.3187-3191.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yamada T., Palm C. J., Brooks B., Kosuge T. Nucleotide sequences of the Pseudomonas savastanoi indoleacetic acid genes show homology with Agrobacterium tumefaciens T-DNA. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6522–6526. doi: 10.1073/pnas.82.19.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  23. Yeh P., Oreglia J., Prévots F., Sicard A. M. A shuttle vector system for Brevibacterium lactofermentum. Gene. 1986;47(2-3):301–306. doi: 10.1016/0378-1119(86)90074-0. [DOI] [PubMed] [Google Scholar]
  24. Yoshihama M., Higashiro K., Rao E. A., Akedo M., Shanabruch W. G., Follettie M. T., Walker G. C., Sinskey A. J. Cloning vector system for Corynebacterium glutamicum. J Bacteriol. 1985 May;162(2):591–597. doi: 10.1128/jb.162.2.591-597.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES