Kinetic and mechanistic studies of morphinone reductase and pentaerythritol tetranitrate reductase

Morphinone reductase from Pseudomonas putida strain M10 and pentaerythritol tetranitrate (PETN) reductase from Enterobacter cloacae strain PB2 are very similar Class I / barrel flavoproteins capable of catalysing biotechnologically relevant reactions. The enzymes have been characterised and conclusions have been drawn about the catalytic and chemical mechanisms of both enzymes.;The kinetics of the reductive half-reaction of both enzymes have been extensively investigated under pre-steady state conditions. Morphinone reductase and PETN reductase form a charge-transfer complex with their preferred reducing substrate (-NADH and -NADPH, respectively) in a second-order rate process. Evidence for an intermediate prior to charge-transfer formation is presented for PETN reductase. Decay of the charge-transfer complex in morphinone reductase and PETN reductase is concomitant with flavin reduction. Thermodynamic analysis of charge-transfer formation and decay, and reduction of the flavin has allowed calculation of an almost complete energy level diagram of the reductive-half-reaction of morphinone reductase.;Four mutant morphinone reductase enzymes were constructed. R238K, R238M and R238E were made to investigate the role of charge near the N1-C2=O2 region of the flavin. Redox potentiometry experiments and pre-steady-state kinetic analyses suggest that positive charge near this region of the flavin is not important in setting the redox potential of the cofactor. The Q104E mutant was made to investigate the role of this residue in the activity of flavin with oxygen. The mutant did not bind flavin, preventing further analysis.