D-2-hydroxyacid Dehydrogenase Research Articles

Methionine Metabolism in Piglets Fed dl-Methionine or Its Hydroxy Analogue Was Affected by Distribution of Enzymes Oxidizing These Sources to Keto-Methionine

Previous evidence shows that the extensive catabolism of dietary essential amino acids (AA) by the intestine results in decreased availability of these AA for protein synthesis in extraintestinal tissues. This raises the possibility that extraintestinal availability of AA may be improved by supplying the animal with an AA source more of which can bypass the intestine. To test this hypothesis, six barrows (35-day-old, 8.6 +/- 1.4 kg), implanted with arterial, portal, and mesenteric catheters, were fed a DL-methionine (DL-MET) or DL-2-hydroxy-4-methylthiobutyrate (DL-HMTB) diet once hourly and infused intramesenterically with 1% p-amino hippurate. Although the directly available L-MET in DL-MET diet was about 1.2-fold that in DL-HMTB diet, the net portal appearance of L-MET was not different between the two diets. Compared with the low mRNA abundance and low activity of D-2-hydroxy acid dehydrogenase (D-HADH) and l-2-hydroxy acid oxidase (L-HAOX) in the intestine, the high mRNA abundance and high activity of D-AA oxidase (D-AAOX) indicated that the intestine had a relatively higher capacity of D-MET utilization than of dl-HMTB utilization to L-MET synthesis and its subsequent metabolism. However, in contrast to the much lower D-AAOX activity (nmol/g tissue) in the stomach than in the liver and kidney, both d-HADH and L-HAOX activity in the stomach was comparable with those in the liver and/or kidney, indicating the substantial capacity of the stomach to convert DL-HMTB to L-MET. Collectively, the difference in distribution of activity and mRNA abundance of D-AAOX, D-HADH, and L-HAOX in the piglets may offer a biological basis for the similar portal appearance of L-MET between DL-MET and DL-HMTB diets, and thus may provide new important insights into nutritional efficiency of different L-MET sources.

Behaviour in different denaturant agents and structural characterization by fluorescence techniques of Haloferax mediterranei D-2-hydroxyacid dehydrogenase

The halophilic enzyme D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei is found to be an oligomeric enzyme composed of two identical subunits. Fluorescence spectra of native and denatured protein and effect of denaturants such as urea and guanidine hydrochloride on enzyme activity of halophilic D-2-hydroxyacid dehydrogenase have been analysed. Native D2-HDH shows the maximum emission at 340nm. The denaturation process caused an exposure to the solvent of the tryptophan residues, as manifested by the red shift of the emission maximum from 340 to 350nm. When urea was used as denaturant agent the enzyme required long incubation times, higher to 24h, to unfold. Fluorescence quenching by KI and acrylamide was also carried out; showing that the tryptophan residues are mainly located near the enzyme surface.

Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei.

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with alpha-ketohexanoic acid and NAD(+) grew under these conditions. Crystals of form I diffracted to beyond 3.0 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 A, beta = 96.3 degrees . Crystals of form II diffracted to beyond 2.0 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 A, alpha = 109.1, beta = 107.5, gamma = 95.9 degrees. The calculated values for V(M) and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry.

Transcription Corepressor CtBP Is an NAD +-Regulated Dehydrogenase

Transcriptional repression is based on the selective actions of recruited corepressor complexes, including those with enzymatic activities. One well-characterized developmentally important corepressor is the C-terminal binding protein (CtBP). Although intriguingly related in sequence to D2 hydroxyacid dehydrogenases, the mechanism by which CtBP functions remains unclear. We report here biochemical and crystallographic studies which reveal that CtBP is a functional dehydrogenase. In addition, both a cofactor-dependent conformational change, with NAD + and NADH being equivalently effective, and the active site residues are linked to the binding of the PXDLS consensus recognition motif on repressors, such as E1A and RIP140. Together, our data suggest that CtBP is an NAD +-regulated component of critical complexes for specific repression events in cells.