Enzymatic synthesis of [4R-2H]NAD (P)H and [4S-2H]NAD(P)H and determination of the stereospecificity of 7 alpha- and 12 alpha hydroxysteroid dehydrogenase.
The stereospecifically labeled coenzymes [4R-2H]NADH, [4R-2H]NADPH and [4S-2H]NAD(P)H were synthesized enzymatically in high yield and high isotopic purity (greater than or equal to 95%) with 2HCOO2H/formate dehydrogenase, (CH3)2C2HOH/alchol dehydrogenase from Thermoanaerobium brockii and [1-2H]glucose/glucose dehydrogenase, respectively. This set of deuterated coenzymes was used to determine the stereospecificity of the previously unstudied 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli (NAD-dependent) and 12 alpha-hydroxysteroid dehydrogenase from Clostridium group P (NADP-dependent). H-NMR and EI-MS of the nicotinamide moiety after enzymatic oxidation of deuterated NAD(P)H with dehydrocholic acid as substrate showed that both dehydrogenases are B-sterospecific.
Ottolina
G
G
Istituto di Chimica degli Ormoni, C.N.R., Milano, Italy.
Riva
S
S
Carrea
G
G
Danieli
B
B
Buckmann
A F
AF
D003013:Clostridium / Q000201:enzymology; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009243:NAD / Q000138:chemical synthesis*; D009249:NADP / Q000138:chemical synthesis*; D013237:Stereoisomerism; D013379:Substrate Specificity
D009243:NAD; D009249:NADP; D003903:Deuterium; D006913:Hydroxysteroid Dehydrogenases