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. 2002 Aug 1;365(Pt 3):731–738. doi: 10.1042/BJ20011714

Support for a three-dimensional structure predicting a Cys-Glu-Lys catalytic triad for Pseudomonas aeruginosa amidase comes from site-directed mutagenesis and mutations altering substrate specificity.

Carlos Novo 1, Sebastien Farnaud 1, Renée Tata 1, Alda Clemente 1, Paul R Brown 1
PMCID: PMC1222709  PMID: 11955282

Abstract

The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

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Selected References

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  1. Ambler R. P., Auffret A. D., Clarke P. H. The amino acid sequence of the aliphatic amidase from Pseudomonas aeruginosa. FEBS Lett. 1987 May 11;215(2):285–290. doi: 10.1016/0014-5793(87)80163-1. [DOI] [PubMed] [Google Scholar]
  2. Arnaud A., Galzy P., Jallageas J. C. Remarques sur l'activité nitrilasique de quelques bactéries. C R Acad Sci Hebd Seances Acad Sci D. 1976 Sep 20;283(5):571–573. [PubMed] [Google Scholar]
  3. Betz J. L., Clarke P. H. Selective evolution of phenylacetamide-utilizing strains of Pseudomonas aeruginosa. J Gen Microbiol. 1972 Nov;73(1):161–174. doi: 10.1099/00221287-73-1-161. [DOI] [PubMed] [Google Scholar]
  4. Bork P., Koonin E. V. A new family of carbon-nitrogen hydrolases. Protein Sci. 1994 Aug;3(8):1344–1346. doi: 10.1002/pro.5560030821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown J. E., Brown P. R., Clarke P. H. Butyramide-utilizing mutants of Pseudomonas aeruginosa 8602 which produce an amidase with altered substrate specificity. J Gen Microbiol. 1969 Aug;57(2):273–285. doi: 10.1099/00221287-57-2-273. [DOI] [PubMed] [Google Scholar]
  6. Brown J. E., Clarke P. H. Mutations in a regulator gene allowing Pseudomonas aeruginosa 8602 to grow on butyramide. J Gen Microbiol. 1970 Dec;64(3):329–342. doi: 10.1099/00221287-64-3-329. [DOI] [PubMed] [Google Scholar]
  7. Brown P. R., Clarke P. H. Amino acid substitution in an amidase produced by an acetanilide-utilizing mutant of Pseudomonas aeruginosa. J Gen Microbiol. 1972 Apr;70(2):287–288. doi: 10.1099/00221287-70-2-287. [DOI] [PubMed] [Google Scholar]
  8. Cheong TK, Oriel PJ. Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherichia coli and marked enhancement of amidase expression using directed evolution*. Enzyme Microb Technol. 2000 Feb 1;26(2-4):152–158. doi: 10.1016/s0141-0229(99)00150-7. [DOI] [PubMed] [Google Scholar]
  9. Farnaud S., Tata R., Sohi M. K., Wan T., Brown P. R., Sutton B. J. Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme. Biochem J. 1999 Jun 15;340(Pt 3):711–714. [PMC free article] [PubMed] [Google Scholar]
  10. Flores T. P., Moss D. S., Thornton J. M. An algorithm for automatically generating protein topology cartoons. Protein Eng. 1994 Jan;7(1):31–37. doi: 10.1093/protein/7.1.31. [DOI] [PubMed] [Google Scholar]
  11. Gomi K., Kitamoto K., Kumagai C. Cloning and molecular characterization of the acetamidase-encoding gene (amdS) from Aspergillus oryzae. Gene. 1991 Dec 1;108(1):91–98. doi: 10.1016/0378-1119(91)90491-s. [DOI] [PubMed] [Google Scholar]
  12. Gregoriou M., Brown P. R. Inhibition of the aliphatic amidase from Pseudomonas aeruginosa by urea and related compounds. Eur J Biochem. 1979 May 2;96(1):101–108. doi: 10.1111/j.1432-1033.1979.tb13018.x. [DOI] [PubMed] [Google Scholar]
  13. Gregoriou M., Brown P. R., Tata R. Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide. J Bacteriol. 1977 Nov;132(2):377–384. doi: 10.1128/jb.132.2.377-384.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  15. Harper D. B. Fungal degradation of aromatic nitriles. Enzymology of C-N cleavage by Fusarium solani. Biochem J. 1977 Dec 1;167(3):685–692. doi: 10.1042/bj1670685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KELLY M., CLARKE P. H. An inducible amidase produced by a strain of Pseudomonas aeruginosa. J Gen Microbiol. 1962 Feb;27:305–316. doi: 10.1099/00221287-27-2-305. [DOI] [PubMed] [Google Scholar]
  17. Karmali A., Pacheco R., Tata R., Brown P. Substitutions of Thr-103-Ile and Trp-138-Gly in amidase from Pseudomonas aeruginosa are responsible for altered kinetic properties and enzyme instability. Mol Biotechnol. 2001 Mar;17(3):201–212. doi: 10.1385/MB:17:3:201. [DOI] [PubMed] [Google Scholar]
  18. Karmali A., Tata R., Brown P. R. Substitution of Glu-59 by Val in amidase from Pseudomonas aeruginosa results in a catalytically inactive enzyme. Mol Biotechnol. 2000 Sep;16(1):5–16. doi: 10.1385/MB:16:1:05. [DOI] [PubMed] [Google Scholar]
  19. Katchalski-Katzir E., Shariv I., Eisenstein M., Friesem A. A., Aflalo C., Vakser I. A. Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2195–2199. doi: 10.1073/pnas.89.6.2195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lesk A. M. CASP2: report on ab initio predictions. Proteins. 1997;Suppl 1:151–166. doi: 10.1002/(sici)1097-0134(1997)1+<151::aid-prot20>3.3.co;2-j. [DOI] [PubMed] [Google Scholar]
  21. Mahenthiralingam E., Draper P., Davis E. O., Colston M. J. Cloning and sequencing of the gene which encodes the highly inducible acetamidase of Mycobacterium smegmatis. J Gen Microbiol. 1993 Mar;139(3):575–583. doi: 10.1099/00221287-139-3-575. [DOI] [PubMed] [Google Scholar]
  22. Marchler-Bauer A., Levitt M., Bryant S. H. A retrospective analysis of CASP2 threading predictions. Proteins. 1997;Suppl 1:83–91. doi: 10.1002/(sici)1097-0134(1997)1+<83::aid-prot12>3.3.co;2-2. [DOI] [PubMed] [Google Scholar]
  23. Martin A. C., MacArthur M. W., Thornton J. M. Assessment of comparative modeling in CASP2. Proteins. 1997;Suppl 1:14–28. doi: 10.1002/(sici)1097-0134(1997)1+<14::aid-prot4>3.3.co;2-f. [DOI] [PubMed] [Google Scholar]
  24. Nakai T., Hasegawa T., Yamashita E., Yamamoto M., Kumasaka T., Ueki T., Nanba H., Ikenaka Y., Takahashi S., Sato M. Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. Structure. 2000 Jul 15;8(7):729–737. doi: 10.1016/s0969-2126(00)00160-x. [DOI] [PubMed] [Google Scholar]
  25. Novo C., Tata R., Clemente A., Brown P. R. Pseudomonas aeruginosa aliphatic amidase is related to the nitrilase/cyanide hydratase enzyme family and Cys166 is predicted to be the active site nucleophile of the catalytic mechanism. FEBS Lett. 1995 Jul 3;367(3):275–279. doi: 10.1016/0014-5793(95)00585-w. [DOI] [PubMed] [Google Scholar]
  26. Ogata K., Umeyama H. Prediction of protein side-chain conformations by principal component analysis for fixed main-chain atoms. Protein Eng. 1997 Apr;10(4):353–359. doi: 10.1093/protein/10.4.353. [DOI] [PubMed] [Google Scholar]
  27. Ogata K., Umeyama H. The role played by environmental residues on sidechain torsional angles within homologous families of proteins: a new method of sidechain modeling. Proteins. 1998 Jun 1;31(4):355–369. [PubMed] [Google Scholar]
  28. Pace H. C., Brenner C. The nitrilase superfamily: classification, structure and function. Genome Biol. 2001 Jan 15;2(1):REVIEWS0001–REVIEWS0001. doi: 10.1186/gb-2001-2-1-reviews0001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pace H. C., Hodawadekar S. C., Draganescu A., Huang J., Bieganowski P., Pekarsky Y., Croce C. M., Brenner C. Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers. 2000 Jul 27-Aug 10Curr Biol. 10(15):907–917. doi: 10.1016/s0960-9822(00)00621-7. [DOI] [PubMed] [Google Scholar]
  30. Peitsch M. C. ProMod and Swiss-Model: Internet-based tools for automated comparative protein modelling. Biochem Soc Trans. 1996 Feb;24(1):274–279. doi: 10.1042/bst0240274. [DOI] [PubMed] [Google Scholar]
  31. Pekarsky Y., Campiglio M., Siprashvili Z., Druck T., Sedkov Y., Tillib S., Draganescu A., Wermuth P., Rothman J. H., Huebner K. Nitrilase and Fhit homologs are encoded as fusion proteins in Drosophila melanogaster and Caenorhabditis elegans. Proc Natl Acad Sci U S A. 1998 Jul 21;95(15):8744–8749. doi: 10.1073/pnas.95.15.8744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rodriguez R., Chinea G., Lopez N., Pons T., Vriend G. Homology modeling, model and software evaluation: three related resources. Bioinformatics. 1998;14(6):523–528. doi: 10.1093/bioinformatics/14.6.523. [DOI] [PubMed] [Google Scholar]
  33. Smyth P. F., Clarke P. H. Catabolite repression of Pseudomonas aeruginosa amidase: the effect of carbon source on amidase synthesis. J Gen Microbiol. 1975 Sep;90(1):81–90. doi: 10.1099/00221287-90-1-81. [DOI] [PubMed] [Google Scholar]
  34. Soubrier F., Lévy-Schil S., Mayaux J. F., Pétré D., Arnaud A., Crouzet J. Cloning and primary structure of the wide-spectrum amidase from Brevibacterium sp. R312: high homology to the amiE product from Pseudomonas aeruginosa. Gene. 1992 Jul 1;116(1):99–104. doi: 10.1016/0378-1119(92)90635-3. [DOI] [PubMed] [Google Scholar]
  35. Stevenson D. E., Feng R., Dumas F., Groleau D., Mihoc A., Storer A. C. Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol Appl Biochem. 1992 Jun;15(3):283–302. [PubMed] [Google Scholar]
  36. THIMANN K. V., MAHADEVAN S. NITRILASE. I. OCCURRENCE, PREPARATION, AND GENERAL PROPERTIES OF THE ENZYME. Arch Biochem Biophys. 1964 Apr;105:133–141. doi: 10.1016/0003-9861(64)90244-9. [DOI] [PubMed] [Google Scholar]
  37. Tata R., Marsh P., Brown P. R. Arg-188 and Trp-144 are implicated in the binding of urea and acetamide to the active site of the amidase from Pseudomonas aeruginosa. Biochim Biophys Acta. 1994 Mar 16;1205(1):139–145. doi: 10.1016/0167-4838(94)90102-3. [DOI] [PubMed] [Google Scholar]
  38. Tomb J. F., White O., Kerlavage A. R., Clayton R. A., Sutton G. G., Fleischmann R. D., Ketchum K. A., Klenk H. P., Gill S., Dougherty B. A. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997 Aug 7;388(6642):539–547. doi: 10.1038/41483. [DOI] [PubMed] [Google Scholar]
  39. Vakser I. A., Aflalo C. Hydrophobic docking: a proposed enhancement to molecular recognition techniques. Proteins. 1994 Dec;20(4):320–329. doi: 10.1002/prot.340200405. [DOI] [PubMed] [Google Scholar]
  40. Vakser I. A. Long-distance potentials: an approach to the multiple-minima problem in ligand-receptor interaction. Protein Eng. 1996 Jan;9(1):37–41. doi: 10.1093/protein/9.1.37. [DOI] [PubMed] [Google Scholar]
  41. Vakser I. A. Low-resolution docking: prediction of complexes for underdetermined structures. Biopolymers. 1996 Sep;39(3):455–464. doi: 10.1002/(SICI)1097-0282(199609)39:3%3C455::AID-BIP16%3E3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  42. Vakser I. A. Protein docking for low-resolution structures. Protein Eng. 1995 Apr;8(4):371–377. doi: 10.1093/protein/8.4.371. [DOI] [PubMed] [Google Scholar]
  43. Xu K., Elliott T. An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium. J Bacteriol. 1993 Aug;175(16):4990–4999. doi: 10.1128/jb.175.16.4990-4999.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yoneda T., Komooka H., Umeyama H. A computer modeling study of the interaction between tissue factor pathway inhibitor and blood coagulation factor Xa. J Protein Chem. 1997 Aug;16(6):597–605. doi: 10.1023/a:1026318823516. [DOI] [PubMed] [Google Scholar]

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