Amino acid substitution at the substrate-binding subsite alters the specificity of the Phanerochaete chrysosporium cellobiose dehydrogenase Desriani, Stefano Ferri, Koji Sode * Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan article info Article history: Received 12 November 2009 Available online 17 December 2009 Keywords: Cellobiose dehydrogenase Cellobiose Lactose Phanerochaete chrysosporium abstract The active site of cellobiose dehydrogenase from Phanerochaete chrysosporium is composed of two subsites, a catalytic C subsite and a substrate-binding B subsite. Based on the crystal structure of the enzyme with a cellobiose analogue, residue Glu279 was selected for site-directed mutagenesis studies. Substitution of Glu279 to Ala, Asn, and Asp had no effect on the expression of the protein in Pichia pastoris but completely abolished its enzymatic activity. Substitution of Glu279 to Gln drastically altered the enzyme’s substrate specificity. While the wild-type cellobiose dehydrogenase efficiently oxidizes cellobi- ose and lactose, the Glu279Gln mutant retained most of its activity with cellobiose but was completely inactive with lactose. We generated structural models of the active site interacting with cellobiose and lactose to provide an interpretation of these results. Ó 2009 Elsevier Inc. All rights reserved. Introduction Cellobiose dehydrogenase (CDH, EC 1.1.99.18) is a fungal extra- cellular hemoflavoprotein that oxidizes cellobiose (Glc-b-1,4-Glc) and other b-1,4-linked disaccharides or oligosaccharides at the C- 1 position to produce the corresponding lactones [1]. The physio- logical role of CDH is to participate, together with other cellulolytic enzymes, in the degradation of cellulose and lignin. CDHs are made up of two domains connected by a serine- and threonine-rich lin- ker [2]. The C-terminal catalytic domain contains a non-covalently bound flavin adenine dinucleotide [3]. The N-terminal cyto- chrome-b-type heme domain transfers electrons from the flavin domain to an external electron acceptor during the oxidation reac- tion. The catalytic domain alone also shows dye-mediated cellobi- ose dehydrogenase activity [4–6]. In addition to its application as a component of cellulolytic en- zymes focusing on biomass conversion, there has recently been intensive investigation on other potential applications of CDH. The enzyme has been used as the anodic catalyst in the construc- tion of an enzyme-fuel cell converting lactose as the substrate [7]. CDH was also employed as a component of a cellobiose enzyme sensor [8,9]. Among the several CDH so far reported, that of the white-rot fungus Phanerochaete chrysosporium has been the most extensively studied, including the separate elucidation of the three-dimen- sional crystal structures of each of the two domains. The active site in the catalytic flavin domain forms a tunnel containing two sub- sites. The reducing sugar moiety binds to the catalytic subsite (C subsite), which has three key amino acid residues for hydrogen bonding, His689, Asn732, and Ser687 [10,11]. The non-reducing sugar moiety binds to the substrate-binding subsite (B subsite), lo- cated near the tunnel entrance, by interacting with Arg586, Glu279, Phe282, and Asn688 [10,12]. CDH has been reported to efficiently oxidize both cellobiose and lactose, which differ only in the orientation at the 4th hydroxyl group of the non-reducing sugar moiety. While the B subsite recog- nizes both glucoside and galactoside moieties of disaccharides, it plays a key role in discriminating against monosaccharides [10]. Previous research has focused on the C subsite amino acid residues, and little is known about the substrate recognition at the B subsite. We recently reported the modification of the substrate specific- ity of galactose/glucose binding protein by a single amino acid sub- stitution [13]. The wild-type protein binds both glucose and galactose with similar binding constants. Substitution of Asp14 to either Asn or Gln had almost no effect on the binding of glucose but almost completely abolished its ability to bind galactose. The modification of galactose/glucose binding protein substrate recog- nition by substituting Asp14, which interacts with the 4th hydro- xyl group of both glucose and galactose, inspired us to attempt a similar investigation on the substrate recognition of CDH. Based on the crystal structure of P. chrysosporium CDH, we introduced amino acid substitutions at the B subsite of the active site to investigate their effects on substrate specificity. We ob- tained one mutant with greatly altered substrate specificity. We created structural models of the active site bound to cellobiose and lactose to help interpret our results. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.12.052 * Corresponding author. Fax: +81 42 388 7027. E-mail addresses: 50006831704@st.tuat.ac.jp (Desriani), stefano@cc.tuat.ac.jp (S. Ferri), sode@cc.tuat.ac.jp (K. Sode). Biochemical and Biophysical Research Communications 391 (2010) 1246–1250 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc