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Structure and kinetics of the beta-lactamase mutants S70A and K73H from Staphylococcus aureus PC1.

Chen CC, Smith TJ, Kapadia G, Wäsch S, Zawadzke LE, Coulson A, Herzberg O
Biochemistry 35 12251-8 (1996)
Related entries: 1djb, 1djc

Cited: 15 times
EuropePMC logo PMID: 8823158

Abstract

Two mutant beta-lactamases from Staphylococcus aureus PC1 which probe key catalytic residues have been produced by site-directed mutagenesis. In the S70A enzyme, the nucleophilic group that attacks the beta-lactam carbonyl carbon atom was eliminated. Consequently, the kcat values for hydrolysis of benzylpenicillin and nitrocefin have been reduced by 10(4)-10(5) compared with the wild-type enzyme. The crystal structure of S70A beta-lactamase has been determined at 2.1 A resolution. With the exception of the mutation site, the structure is identical to that of the native enzyme. The residual activity is attributed either to mistranslation that leads to production of wild-type enzyme and/or to remaining features of the active site that stabilize the tetrahedral transition state. Soaking of the crystals with ampicillin or clavulanate, followed by flash-freezing, has been carried out and the structures examined at 2.0 A resolution. For both experiments, the difference electron density maps revealed buildup of density in the active site that presumably corresponds to beta-lactam binding. However, neither electron density is sufficiently clear for defining the atomic details of the bound compounds. The K73H beta-lactamase has been prepared to test the possible role of Lys73 in proton transfer. It exhibits no detectable activity toward benzylpenicillin, and 10(5)-fold reduction of kcat for nitrocefin hydrolysis compared with the wild-type enzyme. No significant recovery of activity has been measured when the pH was varied between 5.0 and 8.0. The crystal structure of K73H beta-lactamase has been determined at 1.9 A resolution. While the overall structure is similar to that of the native enzyme, the electrostatic interactions between His73 and neighboring residues indicate that the imidazole ring is positively charged. In addition, the hydroxyl group of Ser70 adopts a position that is incompatible with nucleophilic attack on substrates. A crystal soaked with ampicillin was flash-frozen, and diffraction data were collected at 2.1 A resolution. The electron density map showed no indication of substrate binding.

Reviews citing this publication (3)

  1. Catalytic properties of class A beta-lactamases: efficiency and diversity. Matagne A, Lamotte-Brasseur J, Frère JM. Biochem J 330 ( Pt 2) 581-598 (1998)
  2. Molecular analysis of beta-lactamase structure and function. Majiduddin FK, Materon IC, Palzkill TG. Int J Med Microbiol 292 127-137 (2002)
  3. Enzyme catalysis: removing chemically 'essential' residues by site-directed mutagenesis. Peracchi A. Trends Biochem Sci 26 497-503 (2001)

Articles citing this publication (12)

  1. Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme. Nukaga M, Mayama K, Hujer AM, Bonomo RA, Knox JR. J Mol Biol 328 289-301 (2003)
  2. Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme. Zou T, Risso VA, Gavira JA, Sanchez-Ruiz JM, Ozkan SB. Mol Biol Evol 32 132-143 (2015)
  3. Reversible lattice repacking illustrates the temperature dependence of macromolecular interactions. Juers DH, Matthews BW. J Mol Biol 311 851-862 (2001)
  4. The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins. Golemi-Kotra D, Meroueh SO, Kim C, Vakulenko SB, Bulychev A, Stemmler AJ, Stemmler TL, Mobashery S. J Biol Chem 279 34665-34673 (2004)
  5. Adapting the nudged elastic band method for determining minimum-energy paths of chemical reactions in enzymes. Xie L, Liu H, Yang W. J Chem Phys 120 8039-8052 (2004)
  6. Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme. Schneider KD, Bethel CR, Distler AM, Hujer AM, Bonomo RA, Leonard DA. Biochemistry 48 6136-6145 (2009)
  7. Structural and biochemical evidence that a TEM-1 beta-lactamase N170G active site mutant acts via substrate-assisted catalysis. Brown NG, Shanker S, Prasad BV, Palzkill T. J Biol Chem 284 33703-33712 (2009)
  8. Crystallographic Snapshots of Class A β-Lactamase Catalysis Reveal Structural Changes That Facilitate β-Lactam Hydrolysis. Pan X, He Y, Lei J, Huang X, Zhao Y. J Biol Chem 292 4022-4033 (2017)
  9. pKa, MM, and QM studies of mechanisms of beta-lactamases and penicillin-binding proteins: acylation step. Massova I, Kollman PA. J Comput Chem 23 1559-1576 (2002)
  10. Analysis of the binding forces driving the tight interactions between beta-lactamase inhibitory protein-II (BLIP-II) and class A beta-lactamases. Brown NG, Chow DC, Sankaran B, Zwart P, Prasad BV, Palzkill T. J Biol Chem 286 32723-32735 (2011)
  11. Enumerating pathways of proton abstraction based on a spatial and electrostatic analysis of residues in the catalytic site. Chakraborty S. PLoS One 7 e39577 (2012)
  12. Mapping the determinants of catalysis and substrate specificity of the antibiotic resistance enzyme CTX-M β-lactamase. Judge A, Hu L, Sankaran B, Van Riper J, Venkataram Prasad BV, Palzkill T. Commun Biol 6 35 (2023)


Related citations provided by authors (3)

  1. An Engineered Staphylococcus Aureus Pc1 Beta-Lactamase that Hydrolyses Third-Generation Cephalosporins. Zawadzke LE, Smith TJ, Herzberg O Protein Eng. 8 1275- (1995)
  2. Refined Crystal Structure of Beta-Lactamase from Staphylococcus Aureus Pc1 at 2.0. Herzberg O J. Mol. Biol. 217 701- (1991)
  3. Bacterial Resistance to Beta-Lactam Antibiotics. Crystal Structure of Beta-Lactamase from Staphylococcus Aureus Pc1 at 2.5 A Resolution. Herzberg O, Moult J Science 236 694- (1987)

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