Expression, purification, and characterization of Escherichia coli dihydrodipicolinate reductase.
Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carbon-carbon double bond of the alpha,beta-unsaturated cyclic imine dihydrodipicolinate to form the cyclic imine tetrahydrodipicolinate. The enzyme is a component of the bacterial biosynthetic pathway forming L-lysine and diaminopimelate from L-aspartate. The gene encoding dihydrodipicolinate reductase, dapB, has been cloned and sequenced from Escherichia coli (Bouvier et al., 1984), and we have used this sequence information to generate an expression vector containing the dapB gene. Expression and purification of dihydrodipicolinate reductase to homogeneity have allowed us to characterize the kinetics, stereochemistry, and chemical mechanism of the enzymatic reaction. The kinetic mechanism is ordered, with reduced nucleotide binding preceding dihydrodipicolinate binding and presumably with tetrahydrodipicolinate dissociating 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 reductase involving hydride transfer to the beta carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.
Reddy
S G
SG
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
Sacchettini
J C
JC
Blanchard
J S
JS
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D050746:Dihydrodipicolinate Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013237:Stereoisomerism; D013379:Substrate Specificity
D009711:Nucleotides; D010088:Oxidoreductases; D050746:Dihydrodipicolinate Reductase; D044925:Oxidoreductases Acting on CH-CH Group Donors