Stereo-specificity for pro-(R) hydrogen of NAD(P)H during enzyme-catalyzed hydride transfer to CL-20.
A dehydrogenase from Clostridium sp. EDB2 and a diaphorase from Clostridium kluyveri were reacted with CL-20 to gain insights into the enzyme-catalyzed hydride transfer to CL-20, and the enzyme's stereo-specificity for either pro-R or pro-S hydrogens of NAD(P)H. Both enzymes biotransformed CL-20 at rates of 18.5 and 24nmol/h/mg protein, using NADH and NADPH as hydride-source, respectively, to produce a N-denitrohydrogenated product with a molecular weight of 393Da. In enzyme kinetics studies using reduced deuterated pyridine nucleotides, we found a kinetic deuterium isotopic effect of 2-fold on CL-20 biotransformation rate using dehydrogenase enzyme against (R)NADD as a hydride-source compared to either (S)NADD or NADH. Whereas, in case of diaphorase, the kinetic deuterium isotopic effect of about 1.5-fold was observed on CL-20 biotransformation rate using (R)NADPD as hydride-source. In a comparative study with LC-MS, using deuterated and non-deuterated NAD(P)H, we found a positive mass-shift of 1Da in the N-denitrohydrogenated product suggesting the involvement of a deuteride (D(-)) transfer from NAD(P)D. The present study thus revealed that both dehydrogenase and diaphorase enzymes from the two Clostridium species catalyzed a hydride transfer to CL-20 and showed stereo-specificity for pro-R hydrogen of NAD(P)H.
Bhushan
Bharat
B
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Halasz
Annamaria
A
Hawari
Jalal
J
D001372:Aza Compounds / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D047010:Clostridium kluyveri / Q000201:enzymology; D006571:Heterocyclic Compounds / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity
C482139:2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane; D001372:Aza Compounds; D006571:Heterocyclic Compounds; D009249:NADP; D006859:Hydrogen; D010088:Oxidoreductases