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Nuances of Mechanisms and Their Implications for Evolution of the Versatile β-Lactamase Activity: From Biosynthetic Enzymes to Drug Resistance Factors

1997; American Chemical Society; Volume: 119; Issue: 33 Linguagem: Inglês

10.1021/ja963708f

1943-2984

Alexey Bulychev, Irina Massova, Kazuyuki Miyashita, Shahriar Mobashery,

Microbial Natural Products and Biosynthesis

β-Lactamases of classes A and C are believed to have evolved from bacterial enzymes involved in biosynthesis of the peptidoglycan, the so-called penicillin-binding proteins. All these enzymes undergo acylation at an active-site serine by β-lactam antibiotics as a common feature. However, the fate of the acyl-enzyme species is different for β-lactamases and penicillin-binding proteins; deacylation is rapid for the former, whereas it is slow for the latter. It is believed that the acquisition of the ability to deacylate the acyl-enzyme intermediate led to the evolution of β-lactamase activity, which is indispensable for the survival of bacteria in the face of challenge by β-lactam antibiotics. The mechanisms of deacylation of acyl-enzyme intermediates for β-lactamases are examined as a means to investigate structural factors in evolutionary descendency of classes A and C of β-lactamases from penicillin-binding proteins. It is known that in class A β-lactamases the hydrolytic water approaches the acyl-enzyme intermediate from the α-face, a process which is promoted by Glu-166 of these enzymes. An approach from the β-face for class C β-lactamase has been proposed. The process of activation of the hydrolytic water is not entirely understood at the present for these enzymes. Two compounds, p-nitrophenyl (2R,5R)-5-prolylacetate (2) and p-nitrophenyl (1S,3S)-3-carboxycyclopentylacetate (3), were synthesized as mechanistic probes to explore whether the hydrolytic water molecule actually approaches the acyl-enzyme species from the β-face and to investigate a notion that the ring amine at the acyl-enzyme intermediate may promote the hydrolytic reaction. Compound 2 acylates the active site serine of the Q908R β-lactamase (a class C enzyme), and the intermediate undergoes deacylation. On the other hand, compound 3 only acylates the active site, and not having the requisite amine in its structure, the intermediate resists deacylation. Both compounds serve as substrates for the class A TEM-1 β-lactamase, as they were expected, since the approach of the hydrolytic water molecule is from the α-face in this enzyme and is not promoted by the substrate itself. We conclude that substrate-assisted catalysis applies for the class C β-lactamases. On the basis of the evidence discussed, the knowledge of the crystal structures for the classes A and C of β-lactamases and the Steptomyces R61 DD-peptidase/transpeptidase (a PBP), it is proposed herein that evolution of classes A and C of β-lactamases from a primordial penicillin-binding protein should have been independent events; hence, the process does not represent a linear descendency of one β-lactamase from the other.