Rational design of styrene monooxygenase mutants with altered substrate preference

Abstract

Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene derivatives with an a-or b-substituent. Based on the X-ray crystal structure of the oxygenase subunit of styrene monooxygenase, molecular docking of a-ethylstyrene was performed to identify adjacent residues. Four amino acid substitutions (R43A, L44A, L45A, and N46A) were introduced into the enzyme by sitedirected mutagenesis. All four mutations led to a change of substrate preference. The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate a-ethylstyrene.

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

“…LQ26. The investigation of several rationally designed mutants at positions 43-46 leaded to one mutant with substrate preference toward the bulkier substrate (Qaed et al, 2011). However, all of those designed mutants display decreased enzymatic activity, probably as a consequence of reduced FAD binding affinity, because residues 43-46 are located in the center of the putative substrate access channel connecting the postulated styrene and FAD binding cavities and might be involved in FAD binding or could interact with the flavin ring (Feenstra et al, 2006;Ukaegbu et al, 2010).…”

“…The SMO from Pseudomonas sp. LQ26 (designated as StyAB2) was used as the parental enzyme since it has been well studied in our laboratory as a highly selective biocatalyst (Lin et al, 2010(Lin et al, , 2011bQaed et al, 2011), and its high homology with other SMOs from the genus of Pseudomonas would facilitate the docking study. This strategy led to several SMO mutants with increased enzymatic activities toward styrene, and one mutant displayed reversed enantioselectivity toward the substrate 1-phenylcyclohexene.…”

Asymmetric bio-epoxidation catalyzed with the styrene monooxygenase from Pseudomonas sp. LQ26

“…The mutant L45A, in particular, exhibited an altered substrate preference toward the bulkier substrate α-ethylstyrene (Qaed et al 2011). …”

Biocatalysis as an alternative for the production of chiral epoxides: A comparative review

“…SMO also accepts a series of styrene derivatives with substituents on the benzene ring, or at the ␣-or ␤-position of the carbon chain, or with changed aromatic core, resulting in high enantioselectivity in most cases (Schemes 4b, 5c and 6b). However, the enzymatic activity drops significantly for those bearing bulky substituent or electron withdrawing substituent [21,49,50,95,96].…”