Expression, purification, and characterization of escherichia coli dihydrodipicolinate reductase

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Abstract

Dihydrodipicolinate reductase catalyzes the NAD(P)H-dependent reduction of the carbon-carbon double bond of the α,β-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 β carbon of the unsaturated imine. The resonance-stabilized C3 carbanion is protonated to generate the reduced product, tetrahydrodipicolinate.

Original languageEnglish (US)
Pages (from-to)3492-3501
Number of pages10
JournalBiochemistry®
Volume34
Issue number11
StatePublished - 1995

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Dihydrodipicolinate Reductase
Escherichia coli
Imines
Purification
Nucleotides
Carbon
Hydrides
NAD
Enzymes
Stereochemistry
Kinetics
Gene encoding
Biosynthetic Pathways
Reducing Agents
NADP
Aspartic Acid
Genes
Lysine
Hydrogen
Ions

ASJC Scopus subject areas

  • Biochemistry

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Expression, purification, and characterization of escherichia coli dihydrodipicolinate reductase. / Blanchard, John S.

In: Biochemistry®, Vol. 34, No. 11, 1995, p. 3492-3501.

Research output: Contribution to journalArticle

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