The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s. This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

Metabolism of toxic styrene by Pseudomonas putida (S12) bacteria is initiated by a two‐component flavoenzyme system, which consists of the FAD‐specific styrene monooxygenase A (SMOA) and an NADH‐specific styrene monooxygenase B (SMOB), a reductase. Wild‐type SMOB catalyzes the reduction of FAD using a BiBi sequential mechanism and subsequently transfers FAD to the active site of SMOA, which catalyzes the epoxidation of styrene to (S)‐styrene oxide. However, during steady‐state catalysis, we recently discovered that the N‐terminally tagged version, N‐SMOB, employs a double‐displacement mechanism (Kantz, 2005). The efficient delivery of reduced FAD to the SMOA presents a challenge for most two‐component SMO systems and this research aims to further understand the regulation of N‐terminally engineered flavoenzymes.Here we evaluate the impact of an N‐terminal tag on the catalytic mechanisms of SMO. Fluorescence monitored titrations at 4°C determined the Kd value to be ~ 50 nM, an order of magnitude greater than the wild‐type reductase. The increased FAD binding affinity directly affects the steady‐state kinetics at 30°C, as the double‐displacement mechanism with NADH as the leading substrate becomes the preferred method of FAD reduction. Due to these significant changes in flavin binding affinity and catalytic mechanism, we investigated the pre‐steady state and FAD‐transfer kinetics using stopped‐flow fluorescence and absorbance studies. These findings will be presented together with their implications for the engineering of coupled N‐terminally tagged enzymes and N‐terminally linked fusion proteins as potential biocatalysts for the production of essential chiral epoxides.