Dihydrodipicolinate Synthase from E. coli: Characterization of Arginine 138 by Site‐Directed Mutagenesis

Abstract

Dihydrodipicolinate synthase catalyzes the formation of dihydropicolinate from pyruvate and L‐aspartate‐β‐semialdehyde (ASA). The enzyme catalyzes the first committed step for the biosynthesis of L‐lysine in bacteria and plants. The enzyme from Escherichia coli is feedback inhibited by lysine, the end product of the pathway. A study of the pH dependence of the kinetic parameters was done to elucidate the acid‐base chemical mechanism of the enzyme. The kinetic mechanism is ping pong with pyruvate binding to free enzyme. The ε‐amino group of lysine 161 attacks the carbonyl of pyruvate and forms a Schiff base intermediate. The loss of a proton from this intermediate leads to the formation of an enamine intermediate, with the loss of the proton accounting for the irreversible step and the ping pong kinetic mechanism. ASA binds to the enzyme:enamine covalent intermediate. Site‐directed mutagenesis was done to investigate the role of the active site arginine 138. The R138A and R138K mutants were created and the identity of the mutants was confirmed. Kinetic studies will be done to characterize the mutant enzymes.This work was supported by grant P20RR016478 from the National Center for Research Resources a component of the National Institutes of Health and a grant from the University of Central Oklahoma office of Research and Grants to L.C.