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. 1990 Oct 15;271(2):399–406. doi: 10.1042/bj2710399

Role of the conserved amino acids of the 'SDN' loop (Ser130, Asp131 and Asn132) in a class A beta-lactamase studied by site-directed mutagenesis.

F Jacob 1, B Joris 1, S Lepage 1, J Dusart 1, J M Frère 1
PMCID: PMC1149568  PMID: 2173561

Abstract

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

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Selected References

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

  1. Ambler R. P. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci. 1980 May 16;289(1036):321–331. doi: 10.1098/rstb.1980.0049. [DOI] [PubMed] [Google Scholar]
  2. Bush K. Characterization of beta-lactamases. Antimicrob Agents Chemother. 1989 Mar;33(3):259–263. doi: 10.1128/aac.33.3.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bush K. Classification of beta-lactamases: groups 1, 2a, 2b, and 2b'. Antimicrob Agents Chemother. 1989 Mar;33(3):264–270. doi: 10.1128/aac.33.3.264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bush K. Classification of beta-lactamases: groups 2c, 2d, 2e, 3, and 4. Antimicrob Agents Chemother. 1989 Mar;33(3):271–276. doi: 10.1128/aac.33.3.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohen S. A., Pratt R. F. Inactivation of Bacillus cereus beta-lactamase I by 6 beta-bromopencillanic acid: mechanism. Biochemistry. 1980 Aug 19;19(17):3996–4003. doi: 10.1021/bi00558a017. [DOI] [PubMed] [Google Scholar]
  6. Collatz E., Tran Van Nhieu G., Billot-Klein D., Williamson R., Gutmann L. Substitution of serine for arginine in position 162 of TEM-type beta-lactamases extends the substrate profile of mutant enzymes, TEM-7 and TEM-101, to ceftazidime and aztreonam. Gene. 1989 May 30;78(2):349–354. doi: 10.1016/0378-1119(89)90237-0. [DOI] [PubMed] [Google Scholar]
  7. Dalbadie-McFarland G., Cohen L. W., Riggs A. D., Morin C., Itakura K., Richards J. H. Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6409–6413. doi: 10.1073/pnas.79.21.6409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. De Meester F., Joris B., Lenzini M. V., Dehottay P., Erpicium T., Dusart J., Klein D., Ghuysen J. M., Frère J. M., Van Beeumen J. The active sites of the beta-lactamases of Streptomyces cacaoi and Streptomyces albus G. Biochem J. 1987 Jun 1;244(2):427–432. doi: 10.1042/bj2440427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. De Meester F., Joris B., Reckinger G., Bellefroid-Bourguignon C., Frère J. M., Waley S. G. Automated analysis of enzyme inactivation phenomena. Application to beta-lactamases and DD-peptidases. Biochem Pharmacol. 1987 Jul 15;36(14):2393–2403. doi: 10.1016/0006-2952(87)90609-5. [DOI] [PubMed] [Google Scholar]
  10. Dehottay P., Dusart J., Duez C., Lenzini M. V., Martial J. A., Frère J. M., Ghuysen J. M., Kieser T. Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular beta-lactamase from Streptomyces albus G. Gene. 1986;42(1):31–36. doi: 10.1016/0378-1119(86)90147-2. [DOI] [PubMed] [Google Scholar]
  11. Dideberg O., Charlier P., Wéry J. P., Dehottay P., Dusart J., Erpicum T., Frère J. M., Ghuysen J. M. The crystal structure of the beta-lactamase of Streptomyces albus G at 0.3 nm resolution. Biochem J. 1987 Aug 1;245(3):911–913. doi: 10.1042/bj2450911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ellerby L. M., Escobar W. A., Fink A. L., Mitchinson C., Wells J. A. The role of lysine-234 in beta-lactamase catalysis probed by site-directed mutagenesis. Biochemistry. 1990 Jun 19;29(24):5797–5806. doi: 10.1021/bi00476a022. [DOI] [PubMed] [Google Scholar]
  13. Fisher J., Belasco J. G., Khosla S., Knowles J. R. beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. Biochemistry. 1980 Jun 24;19(13):2895–2901. doi: 10.1021/bi00554a012. [DOI] [PubMed] [Google Scholar]
  14. Frère J. M., Dormans C., Duyckaerts C., De Graeve J. Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39. Biochem J. 1982 Dec 1;207(3):437–444. doi: 10.1042/bj2070437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hedge P. J., Spratt B. G. Amino acid substitutions that reduce the affinity of penicillin-binding protein 3 of Escherichia coli for cephalexin. Eur J Biochem. 1985 Aug 15;151(1):111–121. doi: 10.1111/j.1432-1033.1985.tb09075.x. [DOI] [PubMed] [Google Scholar]
  16. Herzberg O., Moult J. Bacterial resistance to beta-lactam antibiotics: crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.5 A resolution. Science. 1987 May 8;236(4802):694–701. doi: 10.1126/science.3107125. [DOI] [PubMed] [Google Scholar]
  17. Hopwood D. A., Kieser T., Wright H. M., Bibb M. J. Plasmids, recombination and chromosome mapping in Streptomyces lividans 66. J Gen Microbiol. 1983 Jul;129(7):2257–2269. doi: 10.1099/00221287-129-7-2257. [DOI] [PubMed] [Google Scholar]
  18. Joris B., Ghuysen J. M., Dive G., Renard A., Dideberg O., Charlier P., Frère J. M., Kelly J. A., Boyington J. C., Moews P. C. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem J. 1988 Mar 1;250(2):313–324. doi: 10.1042/bj2500313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Katz E., Thompson C. J., Hopwood D. A. Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans. J Gen Microbiol. 1983 Sep;129(9):2703–2714. doi: 10.1099/00221287-129-9-2703. [DOI] [PubMed] [Google Scholar]
  20. Leyh-Bouille M., Dusart J., Nguyen-Distèche M., Ghuysen J. M., Reynolds P. E., Perkins H. R. The peptidoglycan crosslinking enzyme system in Streptomyces strains R61, K15 and rimosus. Eur J Biochem. 1977 Nov 15;81(1):19–28. doi: 10.1111/j.1432-1033.1977.tb11922.x. [DOI] [PubMed] [Google Scholar]
  21. Madgwick P. J., Waley S. G. beta-lactamase I from Bacillus cereus. Structure and site-directed mutagenesis. Biochem J. 1987 Dec 15;248(3):657–662. doi: 10.1042/bj2480657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matagne A., Misselyn-Bauduin A. M., Joris B., Erpicum T., Granier B., Frère J. M. The diversity of the catalytic properties of class A beta-lactamases. Biochem J. 1990 Jan 1;265(1):131–146. doi: 10.1042/bj2650131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Moews P. C., Knox J. R., Dideberg O., Charlier P., Frère J. M. Beta-lactamase of Bacillus licheniformis 749/C at 2 A resolution. Proteins. 1990;7(2):156–171. doi: 10.1002/prot.340070205. [DOI] [PubMed] [Google Scholar]
  24. Nguyen-Distèche M., Leyh-Bouille M., Ghuysen J. M. Isolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive D-alanyl-D-alanine-cleaving transpeptidase. Biochem J. 1982 Oct 1;207(1):109–115. doi: 10.1042/bj2070109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oefner C., D'Arcy A., Daly J. J., Gubernator K., Charnas R. L., Heinze I., Hubschwerlen C., Winkler F. K. Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis. Nature. 1990 Jan 18;343(6255):284–288. doi: 10.1038/343284a0. [DOI] [PubMed] [Google Scholar]
  26. Schultz S. C., Richards J. H. Site-saturation studies of beta-lactamase: production and characterization of mutant beta-lactamases with all possible amino acid substitutions at residue 71. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1588–1592. doi: 10.1073/pnas.83.6.1588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sigal I. S., DeGrado W. F., Thomas B. J., Petteway S. R., Jr Purification and properties of thiol beta-lactamase. A mutant of pBR322 beta-lactamase in which the active site serine has been replaced with cysteine. J Biol Chem. 1984 Apr 25;259(8):5327–5332. [PubMed] [Google Scholar]
  28. Sougakoff W., Petit A., Goussard S., Sirot D., Bure A., Courvalin P. Characterization of the plasmid genes blaT-4 and blaT-5 which encode the broad-spectrum beta-lactamases TEM-4 and TEM-5 in enterobacteriaceae. Gene. 1989 May 30;78(2):339–348. doi: 10.1016/0378-1119(89)90236-9. [DOI] [PubMed] [Google Scholar]
  29. Spratt B. G., Cromie K. D. Penicillin-binding proteins of gram-negative bacteria. Rev Infect Dis. 1988 Jul-Aug;10(4):699–711. doi: 10.1093/clinids/10.4.699. [DOI] [PubMed] [Google Scholar]
  30. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]

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