Structural insight into the inactivation of Mycobacterium tuberculosis non-classical transpeptidase LdtMt2 by biapenem and tebipenem.

BACKGROUND: The carbapenem subclass of ß-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of ß-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb). RESULTS: Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (LdtMt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of LdtMt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by ß-lyases. CONCLUSION: The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of LdtMt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.

Defining critical residues for substrate binding to 1-deoxy-D-xylulose 5-phosphate synthase--active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate.

1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate (GraP) in a thiamin diphosphate-dependent manner, and is the first step in the essential pathway to isoprenoids in human pathogens. Understanding the mechanism of this unique enzyme is critical for developing new anti-infective agents that selectively target isoprenoid biosynthesis. The present study used mutagenesis and a combination of protein fluorescence, CD and kinetics experiments to investigate the roles of Arg420, Arg478 and Tyr392 in substrate binding and catalysis. The results support a random sequential, preferred order mechanism, and predict that Arg420 and Arg478 are involved in binding of the acceptor substrate, GraP. D-Glyceraldehyde, an alternative acceptor substrate lacking the phosphoryl group predicted to interact with Arg420 and Arg478, also accelerates decarboxylation of the predecarboxylation intermediate C2α-lactylthiamin diphosphate (LThDP) on DXP synthase, indicating that this binding interaction is not absolutely required, and that the hydroxyaldehyde sufficiently triggers decarboxylation. Unexpectedly, Tyr392 contributes to GraP affinity, and is not required for LThDP formation or its GraP-promoted decarboxylation. Time-resolved CD spectroscopy and NMR experiments indicate that LThDP is significantly stabilized on R420A and Y392F variants as compared with wild-type DXP synthase in the absence of acceptor substrate, but these substitutions do not appear to affect the rate of GraP-promoted LThDP decarboxylation in the presence of high levels of GraP, and LThDP formation remains the rate-limiting step. These results suggest a role of these residues in promoting GraP binding, which in turn facilitates decarboxylation, and also highlight interesting differences between DXP synthase and other thiamin diphosphate-dependent enzymes.