Structural and mechanistic study of antibiotic resistance enzymes: LmDdl2 D-Ala-D-lactate ligase, S130G variant of SHV-1 class A beta-lactamase, and OXA-1 class D beta-lactamase

Date of Completion

January 2005

Keywords

Biophysics, General

Degree

Ph.D.

Abstract

Bacterial resistance to β-lactams and glycopeptide antibiotics is on the rise due to the prevalence of certain antibiotic resistance enzymes. X-ray crystal structures of two β-lactamases (S130G variant of K. pneumoniae SHV-1 class A enzyme, and E. coli OXA-1 class D enzyme) and one ligase (L. mesenteroides LmDdl2 D-Ala-D-lactate ligase) were investigated to help further understanding of the catalytic/substrate profiling mechanisms. ^ The apo (1TDL) and tazobactam complexed (1TDG) S130G mutant SHV-1 structures were the first of a class A β-lactamase without the normally conserved Ser130. Two tazobactam reaction intermediates were identified: one was an unusual out-of-oxyanion-hole acylic enamine species, proposed for a strong remnant tazobactam inactivation. The other was an in-hole aldehyde species, indicating resistance to the inhibitor. A water molecule near the absent Ser130 hydroxyl group helped to maintain the enzymatic proton relay with the same kinact as in the WT SHV-1. Most surprisingly, the expected catalytic water was absent in the apo mutant enzyme and re-emerged in the complex structure accompanied by a catalytic site conformational change. The inhibitor-induced conformational change, as well as the instability of the Ser130 replacing water, might have contributed to the weak binding affinity of the S130G mutant. ^ The OXA-1 class D β-lactamase (1M6K) was found to be monomeric in the crystal, as well as in solution up to 5 mg/ml, with a similar conformation to the dimeric OXA-10. It was carbamylated at pH 7.5 on Lys70. The cavity was highly hydrophobic compared with class A. The hydrophobicity might have helped to lower the pKa of Lys70 value to facilitate the CO2 condensation onto the neutral basic Lys70. Unfortunately, it might also have destabilized the catalytic water in the apo enzyme. ^ The LmDdl2 D-Ala-D-lactate ligase complexed with a phosphoryl phosphinate analog (1EHI) maintained the configurations of the Ω-loop, an active site element, and the key residues Lys260 and Phe261 like the WT D-Ala-D-Ala ligase, even though without a tyrosyl -OH group. The two ligases were similar. Only subtle structural alterations at subsite 2 were noticed i.e. a basic Arg22 at the entry for selectively charge-repelling a zwitterion alanine, a slightly distorted alignment of subsite 2 related to subsite 1, weakened hydrogen bonding and electrostatic interactions on the substrate 2 α-carboxyl terminus, and increased hydrophobicity due to the alteration of Tyr to Phe at 261 plus the inward movement of Met216. The summation of those minor effects might have contributed to the macroscopic selection of D-lactate as a substrate vs D-Ala2. ^

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