On the Importance of a Methyl Group in β-Lactamase Evolution:  Free Energy Profiles and Molecular Modeling

Abstract

beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.

Crystal Structures of Complexes between the R61 DD-peptidase and Peptidoglycan-mimetic β-Lactams: A Non-covalent Complex with a “Perfect Penicillin”

“…This is probably because the active sites of blactamases have selected against extended sidechain D-alanyl-D-alanine peptides. 16,30 Concluding Discussion The suite of structures described here provides insight into the mode of action of a new breed of b-lactam, as well as completing the crystallographic imaging of intermediates in the R61 DDpeptidase reaction mechanism. We have shown that the similar effects of a specific peptidoglycanmimetic side-chain on the reactivity of peptide substrates and b-lactam inhibitors of the R61 DDpeptidase arise from the very similar side-chain a Least-squares fitting was performed with the program XTALVIEW using all atoms in the 16 important active site residues (S62, K65, H108, F120, T123, Y159, N161, D217, W233, Y280, R285, H298, T299, T301, S326, and N327 (146 total atoms)).…”

“…The characteristic D-methyl group of the peptide fits into a snug hydrophobic site adjacent to the side-chain of Trp233. 8,16 If there were a corresponding 6-a-methyl group on the penicillin, it would be directed in such a way as to force the side-chain of Tyr159 out of its position in the E2 structure and thus likely impede its proposed role as a general acid/base in catalysis. 17,18 Strominger and Tipper suggested, on the basis of their structural hypothesis, that 6-a-methyl-penicillins might be better antibiotics than their unsubstituted analogues.…”

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

“…Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, and inhibitor binding [37,42,[55][56][57][58][59]. All of the substrate binding models have made assumptions before the substrate was docked into the active site [37,42], and some of these assumptions have been shown to be invalid for certain substrates [43].…”

Probing the Dynamics of a Mobile Loop above the Active Site of L1, a Metallo-β-lactamase from Stenotrophomonas maltophilia, via Site-directed Mutagenesis and Stopped-flow Fluorescence Spectroscopy

“…Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, protein dynamics, and inhibitor binding (35,(37)(38)(39)(40)(41)(42).…”