A mechanistic study on SMOB-ADP1: an NADH:flavin oxidoreductase of the two-component styrene monooxygenase of Acinetobacter baylyi ADP1.

Two styrene monooxygenase types, StyA/StyB and StyA1/StyA2B, have been described each consisting of an epoxidase and a reductase. A gene fusion which led to the chimeric reductase StyA2B and the occurrence in different phyla are major differences. Identification of SMOA/SMOB-ADP1 of Acinetobacter baylyi ADP1 may enlighten the gene fusion event since phylogenetic analysis indicated both proteins to be more related to StyA2B than to StyA/StyB. SMOB-ADP1 is classified like StyB and StyA2B as HpaC-like reductase. Substrate affinity and turnover number of the homo-dimer SMOB-ADP1 were determined for NADH (24 µM, 64 s(-1)) and FAD (4.4 µM, 56 s(-1)). SMOB-ADP1 catalysis follows a random sequential mechanism, and FAD fluorescence is quenched upon binding to SMOB-ADP1 (K d = 1.8 µM), which clearly distinguishes that reductase from StyB of Pseudomonas. In summary, this study confirmes made assumptions and provides phylogenetic and biochemical data for the differentiation of styrene monooxygenase-related flavin reductases.