Cholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the... Show moreCholera has long been a global concern and in the past decades traditional antibiotic treatments have failed due to the emergence of the antibiotic-resistance of its causative agent, V. cholerae. The resistance is mainly supported by a transmembrane electrochemical gradient of Na+ produced by the respiratory complex Na+-NQR coupled with an internal electron transfer pathway. The assembly and function of Na+-NQR is fulfilled by ApbE, the only known flavin transferase which covalently attaches two FMN molecules to the complex as part of its electron transport chain. Hence, ApbE is closely associated with the cause of antibiotic resistance. Because it does not have any human homologues, ApbE becomes an excellent drug target. In this work, we have investigated the physical properties of the enzyme and clarified its substrate specificity and pH dependence. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on the size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP and potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, pH dependence, mutagenesis, and steady-state kinetic studies have led us to identify the conserved His257 as a residue with dual roles: substrate binding and catalysis. Furthermore, bi-substrate kinetic studies have also revealed that ApbE follows a random Bi Bi mechanism. Together with structural studies, we propose a reaction mechanism where His257 functions as a base, shedding light into the understanding of the ApbE family. Show less
