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Free keywords:
PROTEIN; ASSIGNMENTS; MUTANT; C-13; H-1; ORGANIZATION; INHIBITOR;
TITRATION; RNABiochemistry & Molecular Biology; Physics; Spectroscopy; UVI31+; S55A; beta-lactamases; beta-lactam antibiotics; Sulbactam; NMR;
ITC; MD;
Abstract:
beta-Lactamases (EC 3.5.2.6) confer resistance against beta-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against beta-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative beta-lactamase activity, sulbactam binding (a beta-lactam analogue) in the low mu M affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known beta-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its beta-lactamase activity. Current study is the first report on beta-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for beta-lactamase activity.
