Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.
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-1. 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.
TU Bergakademie Freiberg, Freiberg, Germany.
Thomas
T
Heine
San Francisco State University, San Francisco, CA, USA.
Kathryn
K
Tucker
San Francisco State University, San Francisco, CA, USA.
Nonye
N
Okonkwo
San Francisco State University, San Francisco, CA, USA.
Berhanegebriel
B
Assefa
TU Bergakademie Freiberg, Freiberg, Germany.
Catleen
C
Conrad
TU Bergakademie Freiberg, Freiberg, Germany.
Anika
A
Scholtissek
TU Bergakademie Freiberg, Freiberg, Germany.
Michael
M
Schlömann
San Francisco State University, San Francisco, CA, USA. gassner@sfsu.edu.
George
http://orcid.org/0000-0002-7359-6699
G
Gassner
TU Bergakademie Freiberg, Freiberg, Germany. dirk.tischler@ioez.tu-freiberg.de.|San Francisco State University, San Francisco, CA, USA. dirk.tischler@ioez.tu-freiberg.de.
Dirk
D
Tischler
D055162:Biocatalysis; D001711:Biotransformation; D003001:Cloning, Molecular; D004852:Epoxy Compounds / Q000737:chemistry; D015942:Factor VIIa / Q000235:genetics* / Q000378:metabolism*; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D004852:Epoxy Compounds; D007211:Indoles; D011994:Recombinant Proteins; D009243:NAD; C103587:recombinant FVIIa; D010088:Oxidoreductases; C023111:epoxidase; D010105:Oxygenases; C026721:styrene monooxygenase; D015942:Factor VIIa
Epoxidation; Flavoprotein; Indigo; Monooxygenase; Protein engineering; Styrene