Characterization and Molecular Mechanism of a Novel Cytochrome b 5 Reductase with NAD(P)H Specificity from Mortierella alpina .

The electron-transfer capabilities of cytochrome b 5 reductase (Cyt b 5 R) and NADPH supply have been shown to be critical factors in microbial fatty acid synthesis. Unfortunately, Cyt b 5 R substrate specificity is limited to the coenzyme NADH. In this study, we discovered that a novel Cyt b 5 R from Mortierella alpina ( Ma Cytb5RII) displays affinity for NADPH and NADH. The enzymatic characteristics of high-purity Ma Cytb5RII were determined with the K m, NADPH and K m, NADH being 0.42 and 0.07 mM, respectively. Ma Cytb5RII shows high specific activity at 4 °C and pH 9.0. We anchored the residues that interacted with the coenzymes using the homology models of Ma Cytb5Rs docking NAD(P)H and FAD. The enzyme activity analysis of the purified mutants Ma Cytb5RII [S230N] , Ma Cytb5RII [Y242F] , and Ma Cytb5RII [S272A] revealed that Ser230 is essential for Ma Cytb5RII to have dual NAD(P)H dependence, whereas Tyr242 influences Ma Cytb5RII's NADPH affinity and Ala272 greatly decreases Ma Cytb5RII's NADH affinity.