EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ESCHERICHIA-COLI DIHYDRODIPICOLINATE REDUCTASE

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reductio n of the carbon-carbon double bond of the alpha,beta-unsaturated cycli c imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicol inate. The enzyme is a component of the bacterial biosynthetic pathway forming L-lysine and diaminopimelate from L-aspartate. The gene encod ing dihydrodipicolinate reductase, dapB, has been cloned and sequenced from Escherichia coli (Bouvier et al., 1984), and we have used this s equence information to generate an expression vector containing the da pB gene. Expression and purification of dihydrodipicolinate reductase to homogeneity have allowed us to characterize the kinetics, stereoche mistry, and chemical mechanism of the enzymatic reaction. The kinetic mechanism is ordered, with reduced nucleotide binding preceding dihydr odipicolinate binding and presumably with tetrahydrodipicolinate disso ciating prior to oxidized nucleotide release. The enzyme has a unique nucleotide specificity, with NADH being twice as effective as NADPH as a reductant. The enzyme catalyzes the stereospecific transfer of the 4R hydrogen atom of the reduced nucleotide, as a hydride ion, to the 4 position of dihydrodipicolinate. These results allow us to propose a chemical mechanism for the reaction catalyzed by dihydrodipicolinate r eductase involving hydride transfer to the beta carbon of the unsatura ted imine. The resonance-stabilized C3 carbanion is protonated to gene rate the reduced product, tetrahydrodipicolinate.