Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins

Abstract

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.

Strategies and Methodologies for Developing Microbial Detoxification Systems to Mitigate Mycotoxins

“…Furthermore, the above fusion protein was cloned into a single-vector expression system to couple the epoxidation function to NADH oxidation, thereby enhancing the overall catalytic function of the system. The observed positive changes in the catalytic mechanism were attributed in part to an increased flavin-binding affinity of the StyB reductase associated with its N-terminal extension [160]. In a similar optimization, the NADP(H) dependence of hyperthermophilic 6-phosphogluconate dehydrogenase was engineered to favor the less expensive NAD(H) cofactor for a promising industrial application in bio-batteries [161].…”

A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation

“…Further comparison with a naturally occurring fused SMO system revealed our artificial Fus‐SMO to be about two orders of magnitude more active. Finally, a recent study showed that another artificially fused SMO showed an epoxidation rate more than five times lower than that of our Fus‐SMO . The different behaviour could be attributed to the different types of linkers used for the fusion.…”

“…The styA and styB genes belonging to the bi‐enzymatic system of the SMO from Pseudomonas sp. were genetically fused through a flexible linker, made up of 30 amino acid residues (for details, see Section S4.1 in the Supporting Information). The construct (Fus‐SMO) was designed in the following order: (N‐His 6 ‐tag)‐StyA‐linker‐StyB.…”

“…Additionally, it has been shown that: 1) the highest epoxidation activity is obtained when StyA and StyB are combined at about 1:1 ratio and at low FAD concentration (ca. 15 μ m ),–, and 2) the reduction of oxidised FAD by StyB is the rate‐limiting step . Obtaining a nearly 1:1 ratio mixture of recombinant StyA and StyB in active form in E. coli is still a challenging task.…”

“…Herein we present a chimeric SMO in which reductive (StyB) and epoxidation (StyA) enzymatic units are fused with a flexible linker of 30 amino acids in order: 1) to solve the insolubility issue of StyB, 2) to maximise the epoxidation activity (StyA/StyB 1:1 ratio), and 3) to improve FADH 2 transfer and to find an optimum balance with coupling efficiency (NADH consumption vs. styrene epoxidation). A recent work reported a study on the catalytic mechanisms of other artificially fused SMOs . Nevertheless, these chimeric enzymes were either produced in insoluble (i.e., inactive) form or possessed lower catalytic rates for the epoxidation of styrene (ca.…”