TY - JOUR
T1 - Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase
AU - Reddy, Sreelatha G.
AU - Sacchettini, James C.
AU - Blanchard, John S.
PY - 1995
Y1 - 1995
N2 - Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carboncarbon double bond of the α,α-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 α carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.
AB - Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carboncarbon double bond of the α,α-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 α carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.
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U2 - 10.1021/bi00011a002
DO - 10.1021/bi00011a002
M3 - Article
C2 - 7893644
AN - SCOPUS:0028905536
SN - 0006-2960
VL - 34
SP - 3492
EP - 3501
JO - Biochemistry
JF - Biochemistry
IS - 11
ER -