Built-In Regulatory Mechanisms and Active Site Plasticity Open the Door for Engineering the Styrene Catabolic Pathway

Abstract

Styrene occurs as in the environment as a natural product from the decarboxylation of cinnamic acid and the incomplete combustion of organic material. In recent times increased reliance on the combustion of fossil fuels, and the industrial synthesis of styrene for the production of plastic and rubber products has resulted in dramatic increases in environmental exposure and contamination. Microorganisms evolved from styrene-contaminated environments have developed strategies for detoxification and catabolism of styrene that provide insight for the development of strategies in environmental remediation and biocatalysis.Pseudomonas bacteria employ a styrene-induced three-step enzymatic pathway initiated by the enantioselective epoxidation of styrene to S-styrene oxide by styrene monooxygenase (SMO), a two-component flavoprotein composed of the FAD-specific styrene epoxidase SMOA and NADH-specific flavin reductase, SMOB. Styrene oxide is then isomerized to phenylacetaldehyde by styrene oxide isomerase (SOI), and finally oxidized to phenylacetic acid by an NAD-dependent phenylacetaldehyde dehydrogenase (PADH).In the present work we evaluate the role valine side chains in the catalytic active site of SMOA at positions 211 and 303 in defining the catalytic specificity and efficiency of SMO. Single-turnover stopped-flow fluorescence studies of alanine and isoleucine mutations at these sites indicate that changes in steric bulk primarily impacts the kinetics of the epoxidation reaction with little impact on the subsequent kinetics of elimination of water from the C(4a)-hydroxyflavin intermediate.The styrene metabolic pathway is dependent of the exchange of FAD between the reductase and epoxidase components of SMO and of pyridine nucleotide between the reductase and dehydrogenase. Regulatory reductase-epoxidase and reductase-dehydrogenase interactions will be presented that allow this pathway to be engineered to accommodate a broad range of substrate epoxidation rates while remaining catalytically efficient.