PubMed · 9590080
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Y Ogawa, T Hatano. 1998. [Primary hyperoxaluria type 2(PH2)].. https://pubmed.ncbi.nlm.nih.gov/9590080/
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Reduction of 2-oxoacids to the corresponding (S)-2-hydroxyacids is an important transformation in biochemistry. To date all (S)-2-hydroxyacid dehydrogenases belonging to the L-lactate/L-malate dehydrogenase family have been found to transfer the pro-4R hydrogen of either NADH or NADPH to C-2 of the 2-oxoacid substrates during their reduction. Here, we report that recombinantly generated (S)-2-hydroxyacid dehydrogenases present in the methanoarchaea Methanococcus jannaschii and Methanothermus fervidus use the pro-4S hydrogen of NADH to reduce a series of 2-oxoacids to the corresponding (S)-2-hydroxyacids. This information as well as the low sequence identity between these archaeal enzymes and the L-lactate/L-malate family of enzymes indicate that these enzymes are not evolutionary related and therefore constitute a new class of (S)-2-hydroxyacid dehydrogenases.
Pig aldehyde reductase containing the active site mutation tyrosine(50) to phenylalanine has been crystallized in the presence of the cofactor NADP(H) to a resolution of 2.2 A. This structure clearly shows loss of the tyrosine hydroxyl group and no other significant perturbations compared with previously determined structures. The mutant binds cofactor (both oxidized and reduced) more tightly than the wild-type enzyme but shows a complete lack of binding of the aldehyde reductase inhibitor barbitone, as determined by fluorescence titrations. Numerous attempts at preparing a ternary complex with a range of small aldehyde substrates were unsuccessful. This result, in addition to the inability of the mutant protein to bind the inhibitor, provides strong evidence for the proposal that the tyrosine hydroxyl group is essential for substrate binding in addition to catalysis.
A 1.9 A resolution X-ray structure of the apo-form of Corynebacterium 2,5-diketo-d-gluconic acid reductase A (2,5-DKGR A), a member of the aldo-keto reductase superfamily, has been determined by molecular replacement using the NADPH-bound form of the same enzyme as the search model. 2,5-DKGR A catalyzes the NADPH-dependent stereo-specific reduction of 2,5-diketo-d-gluconate (2,5-DKG) to 2-keto-l-gulonate, a precursor in the industrial production of vitamin C. An atomic-resolution structure for the apo-form of the enzyme, in conjunction with our previously reported high-resolution X-ray structure for the holo-enzyme and holo/substrate model, allows a comparative analysis of structural changes that accompany cofactor binding. The results show that regions of the active site undergo coordinated conformational changes of up to 8 A. These conformational changes result in the organization and structural rearrangement of residues associated with substrate binding and catalysis. Thus, NADPH functions not only to provide a hydride ion for catalytic reduction, but is also a critical structural component for formation of a catalytically competent form of DKGR A.