Development of a satisfactory and general continuous assay for aminotransferases by coupling with (R)-2-hydroxyglutarate dehydrogenase.

A continuous general spectrophotometric assay for measuring the activity of aminotransferases has been developed. It is based on the transamination of a keto compound (amino acceptor) and l-glutamate (amino donor), yielding the corresponding amino compound and 2-oxoglutarate. The rate of formation of 2-oxoglutarate is measured in a coupled reaction with overproduced recombinant nicotinamide adenine dinucleotide (NAD(+))-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans, with the rate of absorbance decrease at 340nm indirectly reflecting the aminotransferase activity.

Structural basis for stereo-specific catalysis in NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans.

NAD(+)-dependent (R)-2-hydroxyglutarate dehydrogenase (HGDH) catalyses the reduction of 2-oxoglutarate to (R)-2-hydroxyglutarate and belongs to the d-2-hydroxyacid NAD(+)-dependent dehydrogenase (d-2-hydroxyacid dehydrogenase) protein family. Its crystal structure was determined by phase combination to 1.98 A resolution.