ORIGINAL RESEARCH PAPER Rational design of styrene monooxygenase mutants with altered substrate preference Abeer Ahmed Qaed • Hui Lin • De-Fang Tang • Zhong-Liu Wu Received: 13 October 2010 / Accepted: 3 November 2010 / Published online: 19 November 2010 Ó Springer Science+Business Media B.V. 2010 Abstract Styrene monooxygenase catalyzes the enantioselective epoxidation of styrene but displays significantly decreased activity toward styrene deriv- atives 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 site- directed 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. Keywords Biocatalysis Á Protein engineering Á Rational design Á Styrene monooxygenase Á Substrate preference Introduction Styrene monooxygenase (SMO) is a two-component flavoenzyme composed of a flavin adenine dinucleotide (FAD)-specific styrene epoxidase, StyA (the larger oxygenase domain) that catalyzes the epoxidation of C = C double bonds, and an NADH-specific flavin reductase, StyB (the smaller reductase domain) that catalyzes the two-electron reduction of FAD (Otto et al. 2004; Kantz et al. 2005). It is a highly enantioselective enzyme that catalyzes the formation of (S)-styrene oxide from styrene with an excellent enantiomeric excess of more than 99% ee (Bernasconi et al. 2000; Panke et al. 2000; Bernasconi et al. 2004; Han et al. 2006), which provides an alternative to the generally applied Sharp- less-Katsuki epoxidation (Sharpless 2002) and Jacobsen epoxidation (Jacobsen et al. 1991) for the synthesis of chiral building blocks for pharmaceuticals or agro- chemicals (Noyori et al. 2003). The SMO from Pseu- domonas fluorescens VLB120 has been successfully used in the pilot scale production of (S)-styrene oxide (Panke et al. 2002). In the study of the bioconversion of styrene derivatives, significant steric hindrance has been observed when the substrate carries an a- or b-substituent (Bernasconi et al. 2000; Schmid et al. 2001). This behavior is common for enzymatic reactions, and considerable effort has been made to change the substrate bias of enzymes (Rubin-Pitel and Zhao 2006; Ema et al. 2010; Wu et al. 2005). Protein engineering of SMO was undertaken using a random-library screening approach based on the model reaction of indigo formation and generated SMO variants with improved activity (Gursky et al. 2010). However, the same screening method cannot A. A. Qaed Á H. Lin Á D.-F. Tang Á Z.-L. Wu (&) Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, China e-mail: wuzhl@cib.ac.cn A. A. Qaed Á H. Lin Graduate University of the Chinese Academy of Sciences, Beijing 100049, China 123 Biotechnol Lett (2011) 33:611–616 DOI 10.1007/s10529-010-0472-9