Transition State Analysis of the α-Carboxyketose Synthases NeuB and DAHP Synthase

Abstract

Antibiotic resistance is a global public health challenge. On current trends, antibiotic-resistant infections could cause an additional 10 million deaths per year by 2050, surpassing cancer. New antimicrobial compounds are desperately needed. The α-carboxyketose synthases (αCKSs) are antibiotic targets that catalyze aldol-like reactions of phosphoenolpyruvate (PEP) and aldoses to form sugars essential to bacterial survival or virulence. These include 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) (amino acid synthesis) and a sialic acid synthase, NeuB (immune evasion). More than 40 years of inhibitor design has yielded few potent inhibitors and no rational basis for improving them. We have performed transition state (TS) analysis on NeuB and DAHPS using multiple kinetic isotope effects (KIEs) to reveal the TS structure and provide a target for rational inhibitor design. We developed new methods of KIE measurement by NMR and measured the 3-13C-, 2-13C-, and 2-18O KIEs for NeuB and DAHPS. For NeuB, the 3-13C KIE is normal, while the 2-13C, and 2-18O KIEs are close to unity. This reveals that the first irreversible transition state is for formation of the tetrahedral intermediate (THI), rather than its breakdown and that the TS involves formation of the C3∙∙∙C1’ bond with no involvement of the water nucleophile, and little charge development. This TS structure explains the ineffectiveness of previous cationic inhibitors and suggests a route forward for inhibitor design. For DAHPS, the 3-13C KIE is normal; the 2-18O KIE is close to unity, and the 2-13C KIE is inverse. The DAHPS TS is therefore similar to NeuB’s, with similar implications for inhibitor design, but further optimization is needed to fully understand the inverse KIE at C2.