A kinetic and spectroscopic study of tetrahydrodipicolinate N-succinyltransferase (DapD) from Serratia marcescens and its inactivation by Cu2+

Abstract

Tetrahydrodipicolinate N-succinyltransferase (DapD) catalyzes the reaction of tetrahydrodipicolinate (THDP) and succinyl-CoA to form (S)-2-(3-carboxypropanamido)-6-oxoheptanedioic acid and coenzyme A. The enzyme is in the diaminopimelate-lysine biosynthesis pathway which produces two metabolites necessary for the survival and growth of pathogenic bacteria. Since lysine is an essential amino acid to humans, DapD is a potentially safe target for antibiotic therapies. Despite its identification as an exploitable target, details regarding the mechanism of DapD and its inhibition remain poorly resolved. In this work, the DAPD gene from Serratia marcescens has been optimally expressed in E. coli host cells and purified. Initial velocity patterns generated using two assays (one direct and one coupled) are consistent with a rapid equilibrium ordered bi bi kinetic mechanism. Therefore, conversion of the central enzyme complexes (chemistry) is thought to be the rate-limiting step in the reaction. Data fitted to the appropriate rate equation provide estimates of kinetic constants for the reaction (K_ia (2-AP) = 1.9 ± 0.26 mM and K_b (succinyl-CoA) = 87 ± 15 μM). SmDapD is rapidly and specifically inactivated in the presence of Cu²⁺ at low concentrations, yet activity was protected in a reducing environment (1 mM DTT). Correspondingly, the internal tryptophan fluorescence of DapD is quenched by Cu²⁺ with a K_D of 2.7 μM, and emission spectra are comparable to chemically denatured DapD. Fluorescence quenching was relieved in the presence of DTT or EDTA. EPR spectra are consistent with Cu²⁺ binding to enzymatic histidine and being reduced to Cu⁺. These data suggest Cu²⁺ binding to the enzyme as well as an oxidative inactivation that results in significant structural changes and denaturing. Together, the mechanistic detail and characterization of the Cu²⁺ inactivation will inform the targeting of DapD with new antibiotic therapies.