Expression, Purification, and Characterization of Escherichia coli Dihydrodipicolinate Reductase

Abstract

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.

Enzymology of Bacterial Lysine Biosynthesis

“…The enzyme functions by utilising either phosphorylated or non-phosphorylated pyridine nucleotides, NAD(P)H, as hydrogen donors to carry out its reaction. The kinetic mechanism of E. coli DHDPR is ordered and sequential (Reddy et al, 1995), involving binding of NAD(P)H followed by DHDP. The reaction is initiated by hydride transfer from the 4-pro-R position of NAD(P)H to the C4-position of DHDP, with the resultant enamine then undergoing tautomerisation to form THDP.…”

“…The reaction is initiated by hydride transfer from the 4-pro-R position of NAD(P)H to the C4-position of DHDP, with the resultant enamine then undergoing tautomerisation to form THDP. Upon completion of the reaction, the release of the product THDP is followed by NAD(P) + release (Reddy et al, 1995) (Fig. 7).…”

“…1). HTPA is non-enzymatically dehydrated to produce dihydrodipicolinate (DHDP), which is subsequently reduced by the NAD(P)H-dependent enzyme, dihydrodipicolinate reductase (DHDPR, EC 1.3.1.26), to form L-2,3,4,5,-tetrahydrodipicolinate (THDP) (Dommaraju et al, 2011;Girish et al, 2011;Reddy et al, 1995 (Fig. 1).…”

Two complete syntheses of (S)-aspartate semi-aldehyde and demonstration that Δ2-tetrahydroisophthalic acid is a non-competitive inhibitor of dihydrodipicolinate synthase

“…3,4 This method is still commonly used today, presumably due to the one step procedure from allylglycine. [10][11][12] However, studies in our laboratory using the DHDPS/DHDPR coupled enzymatic assay 7 have suggested that the ozonolysis product is contaminated by material that has an inhibitory effect on the DHDPS enzyme. 13 To address the deficiencies of the Black and Wright method, Tudor and coworkers have investigated the ozonolysis of (S)-N-tert-butoxycarbonyl allyglycine p-methoxybenzyl ester.…”

Cloning of the dapB gene, encoding dihydrodipicolinate reductase, from Mycobacterium tuberculosis

“…1) (9). The DHPR protein of E. coli functions as a tetramer and shows an unusual ability to utilize either NADH or NADPH as a cofactor for catalysis, with the former nucleotide only slightly preferred over the latter (20). We do not know which variant DAP pathway is used by M. tuberculosis; therefore, we directed our attention to the dapB gene, since the DHPR-catalyzed reaction yields the substrate for all three variant DAP biosynthetic pathways (Fig.…”