glyoxylate reductase

Metode spectofotometrice de identificare a enzimei:The Paracoccus sp. 12-A FDHThe activity of FDHs toward formate was assayed at 30 _C in 100 mM sodium phosphate buffer (pH 7.0) containing 1.0 mM NAD+ and various concentrations of sodium formate. The activity of FDHs toward glyoxylate was assayed at 30 _C in 50 mM sodium MES buffer (pH 5.5) containing 0.1 mM NADH and various concentrations of glyoxylate. One unit was defined as the conversion of 1 lmol of substrate per 1 min. Kinetic parameters were calculated from plots of v/[S] versus[S]. The deuterium derivative of formic acid was purchased from Sigma–Aldrich, and used for determination of the primary isotope effects on glyoxylate reduction of the wild-type and mutant FDHs. Protein concentrations were calculated using an extinction coefficients at 280 nm of 47,330M_1 cm_1 for FDHs, as determined from the amino acid composition and molecular weight of Paracoccus sp. 12-A FDH .

Based on the genome sequence of Rhizobium etli, the nodulating endosymbiont of the common bean plant, wepredicted a putative 3-phosphoglycerate dehydrogenase to exhibit GHPR activity instead. The protein was overexpressed and purified. The enzyme ishomodimeric under native conditions and is indeed capable of reducing both glyoxylate and hydroxypyruvate. Other substrates are phenylpyruvate andketobutyrate. The highest activity was observed with glyoxylate and phenylpyruvate, both having approximately the same kcat/Km ratio. This kind ofsubstrate specificity has not been reported previously for a GHPR. The optimal pH for the reduction of phenylpyruvate to phenyllactate is pH 7. Thesedata lend support to the idea of predicting enzymatic substrate specificity based on phylogenetic clustering.The assay mixture was incubated for 15 min. 50 μl from this reactionmixture was analyzed on a BioRad Aminex HPX-87H (300 mm×7.8 mm)column by a high performance liquid chromatograph (HPLC) (Spectra PhysicsSP8800) equipped with a variable wavelength UV-visible detector (SP8450).The mobile phase was 6 mM HCl (pH 2). The flow rate was 0.6 ml min−1, andthe eluate was monitored at 210 nm.

An alternate spectrophotometric assay for glyoxylate was developed by Zarembski andHodgkinson in 1965 , based on the colored product produced by the reaction ofglyoxylate and resorcinol in the presence of an acid extract of Psuedomonas oxalaticus.The glyoxylate : rescorcinol adduct is spectrophotometrically visible with a λmax of490nm, and detection limit of thirteen micro-molar. In addition the colored product isalso visible by fluorescence within a pH range of 7 to 9. This procedure while notexplicitly stated in the reference is dependent upon the enzymatic activity of glyoxylatedehydrogenase which is a metabolic product of the growth of Psuedomonas only underhigh oxalate conditions. The Zarembski method is reliant on the oxalate supportedPsuedomonas extract, an extract which is extremely time and preparation intensive toproduce in any appreciable quantities.

Figure 1. Spectrophotometric assays for glyoxylate. Overview of the spectrophotometric assays developed for the analysis of glyoxylate. (A) The malate synthase / malate dehydrogenase assay, detection is based upon the concomitant reduction of NADH and oxidation PMS to yield a tetrazolium dye. (B) Theglycolate oxidase assay for the stoichiometric production hydrogen peroxide based on glyoxylate consumption. (C) The glyoxylate reductase dependant glyoxylate reduction upon NADPH oxidation visible at 340nm.The enzyme-coupled assay for glyoxylate was initiated by the addition of malate synthaseand malate dehydrogenase. The assay contained a standard solution of 100 mM TEA-HClpH 7.8, 150μM / 8.25μM MTS/PMS, 10mM MgCl2, 400μM acetyl-CoA, 500μM NAD+,0-50μM glyoxylate, 6U/mL malate synthase, and 6U/mL malate dehydrogenase in a finalvolume of 1mL. The absorbance at 490 nm was measured after 1 hr incubation at 37° Cin the dark. The small amount of MTS reduced for the zero glyoxylate control wassubtracted from that obtained in the presence of glyoxylate.

This cytosolic enzyme plays a role in the metabolism of glyoxylate and in the production of gluconeogenic precursors from serine via hydroxypyruvate. In PH2 glyoxylate and hydroxypyruvate are metabolised to oxalate and L-glycerate respectively, in reactions believed to be catalysed by lactate dehydrogenase (LDH). This accumulation of L-glycerate and excretion into the urine has been regarded as pathognomonic for PH2 ever since the first description of the disease in 1968. However, two cases have recently been described where L-glycericaciduria was not a prominent feature suggesting that the disease may be mis - or under-diagnosed. Certainly there is evidence that groups ofhyperoxaluric patients may include some PH2 patients misdiagnosed as PH1 on the basis of disease severity.Crude extracts were purified by nickel affinity column , eluting recombinant GRHPR with 500mM imidazole. Purified protein was dialysed overnight in T3 dialysis membrane against 20mM potassium phosphate buffer, pH7.0 and quantified by absorbance at 280nm.

Bibliografie

Shinoda T., Arai K., Taguchi H., 2007. A highly specific glyoxylate reductase derived from a formate dehydrogenase.

Fauvart M., Braeken K., Daniels R., Vos K., Ndayizeye M., Noben J.P., Robben J., Vanderleyden J., Michiels J., 2007. Identification of a novel glyoxylate reductase supports phylogeny-based enzymatic substrate specificity prediction.

Carpenter S.E., 2006. Enzyme linked spectroscopic assays for Glyoxylate:The use of Peptidylglycine alpha-Amidating Monoxygenase for the discovery of Novel alpha-Amidated hormones.

D. P. Cregeen, E.L. Williams, S. Hulton, G. Rumsby, 2003. Molecular Analysis of the Glyoxylate Reductase (GRHPR) Gene and Description of Mutations Underlying Primary Hyperoxaluria Type 2