Short Communication A combined approach of experiments and computational docking simulation to the Coprinus cinereus peroxidase-catalyzed oxidative polymerization of alkyl phenols Jong Chul Park a,1 , Jeong Chan Joo a,1 , Eun Suk An c , Bong Keun Song c , Yong Hwan Kim b,⇑ , Young Je Yoo a,⇑ a School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea b Department of Chemical Engineering, Kwangwoon University, Seoul 139-701, Republic of Korea c Korea Research Institute of Chemical Technology, Taejon, Republic of Korea article info Article history: Received 1 June 2010 Received in revised form 1 December 2010 Accepted 3 December 2010 Available online 9 December 2010 Keywords: Alkyl phenols Coprinus cinereus peroxidase (CIP) Oxidative polymerization Substrate specificity Computational docking simulation abstract The characteristics of the oxidative polymerization of alkyl phenol derivatives catalyzed by Coprinus cinereus peroxidase (CIP) were studied qualitatively and quantitatively using a combined approach of experiments and computational docking simulations. As determined by docking study of CIP and alkyl phenols, the binding interaction was found to be important for the determination of substrate specificity. The distant binding and indirect orientation of o-isopropyl phenol and o-tertiary butyl phenol to the catalytic residue (56His) could explain the inability of CIP to polymerize these substrates. Three hydrophobic residues (156Pro, 192Leu, and 230Phe) at the entrance of the binding pocket were also found to be crucial in binding and orientation of alkyl phenols. A two-parameter QSAR equation with the binding distance and the molecular volume of the substrates was proposed and the polymerization yield was accurately predicted by two-parameter QSAR equation. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction The peroxidases including Horseradish peroxidase (HRP), Soybean peroxidase (SBP), and CIP can polymerize various alkyl phenols but CIP is much versatile due to better reaction rate and broad substrate specificity toward alkyl phenols (Ikehata et al., 2005; Kim et al., 2005). This phenomenon might result from differ- ent enzyme-substrate interactions in the active site but few efforts have sought to understand the polymerization characteristics of peroxidases at the molecular level (Ziemys and Kulys, 2005). In particular, despite its industrial importance, there is no systematic understanding of the effect of substrate’s structure on the charac- teristics of the oxidative polymerization catalyzed by peroxidases such as substrate specificity or polymerization yield. In this study, we performed a combined approach of experi- ments and computational docking to systematically elucidate the effect of substrate’s structure on the polymerization characteristics of CIP such as substrate specificity and polymerization yield. In qualitative analysis, the substrate specificity of CIP could be governed by the specific interactions between alkyl phenols and CIP, i.e., close binding distance and direct orientation, rather than the chain length of substrate or the size of binding pocket. More- over, the chemical property of the binding pocket revealed that three residues (156Pro, 192Leu, and 230Phe) have an important role in binding alkyl phenols and the orientation of the reactive OH group. In quantitative analysis, a two-parameter QSAR equa- tion for oxidative polymerization of alkyl phenols could be pro- posed by using the binding distance between the reactive OH group of the substrates and nitrogen atom of the catalytic residue 56His (d His ) and molecular volume of substrate (V M ), which could accurately predict the experimental yield. This combined study for 13 alkyl phenols polymerization could be used as a preliminary tool to select favorable substrates for oxidative polymerization of other peroxidases, as well as a practi- cal tool to design the CIP variants to polymerize nonpolymerizable alkyl phenols by mutating 156Pro, 192Leu, and 230Phe. Moreover, recent peroxidase study (Cho et al., 2009) revealed that CIP could improve microbial production of butanol by removing phenolic compounds in lignocellulosic hydrolysates which cause inhibitory effects on cell growth and butanol production. Therefore, our computational study would be used to design promising CIP mutants with a broad substrate specificity or better polymerization activity toward phenolic compounds, which could remove the phenolic inhibitors more effectively and lead to more butanol production. 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.021 ⇑ Corresponding authors. Tel.: +82 2 880 7411; fax: +82 2 887 1659 (Y.J. Yoo), tel.: +82 2 940 5675 (Y.H. Kim). E-mail addresses: metalkim@kw.ac.kr (Y.H. Kim), yjyoo@snu.ac.kr (Y.J. Yoo). 1 These authors equally contributed to this work. Bioresource Technology 102 (2011) 4901–4904 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech