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Engineering New Substrate Specificity into the Active Site of Styrene Monooxygenases
Author(s) -
Truong Phu,
Gassner George
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.572.3
Subject(s) - active site , styrene , monooxygenase , chemistry , substrate (aquarium) , mutagenesis , directed evolution , stereochemistry , steric effects , protein engineering , mutant , biochemistry , combinatorial chemistry , enzyme , organic chemistry , cytochrome p450 , copolymer , biology , ecology , gene , polymer
Styrene Monooxygenases catalyze the FAD‐dependent epoxidation of styrene to (S)‐styrene oxide in the first step of the styrene catabolic and detoxification pathways employed by a range of microorganisms. The robust tolerance of the styrene‐binding pocket to mutagenesis and the tunability of the active site chemistry of SMOs has identified these enzymes a valuable new target for development as biocatalysts with applications in chiral synthesis and bioremediation. In the present study, we systematically evaluate the impact of changing hydrophobic volume in the styrene‐binding pocket of an N‐terminally histidine‐tagged epoxidase (NSMOA) by using the active site‐directed mutants, V211A, V303A, V211I, and V303I. We find the equilibrium midpoint potential of FAD bound to these mutants to be indistinguishable from the wild‐type protein, indicating that the electronic environment of the FAD is not significantly changed by the amino acid substitutions. The kinetics of the epoxidation reaction of a library of substrate analogs were monitored by single‐turnover studies by stopped‐flow fluorescence spectroscopy. Evaluation of the kinetic results by sequential exponential fitting indicates that the substrate specificity of NSMOA (V303A) shifts to favor epoxidation of the sterically‐bulky substrate 4‐vinylbiphenyl over styrene by 1.5 fold. We conclude that active site directed mutations that change the hydrophobic volume in the active site of NSMOA provide a direct means of tuning the substrate specificity without perturbing the electronic properties of the FAD.