Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect β-N-acetyl-D-hexosaminidase

et al. (2012) Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect b-N- acetyl-D-hexosaminidase. PLoS ONE 7(12): e52225. doi:10.1371/journal.pone.0052225 Structural Insights into Cellulolytic and Chitinolytic Enzymes Revealing Crucial Residues of Insect b-N-acetyl- D-hexosaminidase Tian Liu 0 Yong Zhou 0 Lei Chen 0 Wei Chen 0 Lin Liu 0 Xu Shen 0 Wenqing Zhang 0 Jianzhen Zhang 0 Qing Yang 0 Daniel Doucet, Natural Resources Canada, Canada 0 1 School of Life Science and Biotechnology, Dalian University of Technology , Dalian , China , 2 School of Software Technology, Dalian University of Technology , Dalian , China , 3 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai , China , 4 State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University , Guangzhou , China , 5 Research Institute of Applied Biology, Shanxi University , Taiyuan , China The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind b1,4-linked glucose residues in a similar manner. A structural and mutational analysis was performed for the plant cellulolytic enzyme BGlu1 from Oryza sativa and the insect chitinolytic enzyme OfHex1 from Ostrinia furnacalis. Although BGlu1 shows little amino-acid sequence or topological similarity with OfHex1, three residues (Trp490, Glu328, Val327 in OfHex1, and Trp358, Tyr131 and Ile179 in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu328 together with Trp490 was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in Km for (GlcNAc)2 and a 42-fold increment in Ki for TMG-chitotriomycin. A crystal structure of E328A in complex with TMGchitotriomycin was resolved at 2.5 A , revealing the obvious conformational changes of the catalytic residues (Glu368 and Asp367) and the absence of the hydrogen bond between E328A and the C3-OH of the +1 sugar. V327G exhibited the same activity as the wild-type, but acquired the ability to efficiently hydrolyse b-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu328 and Val327 were identified as important for substrate-binding and as glycosidic-bond determinants. A structurebased sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes. - Funding: The authors acknowledge the financial support provided by the National Key Project for Basic Research (2010CB126100), the National Natural Science Foundation of China (31070715, 31101671), the National High Technology Research and Development Program of China (2011AA10A204), the National Key Technology R&D Program (2011BAE06B05), and the Fundamental Research Funds for the Central Universities (DUT11ZD113, DUT11RC(3)73). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cellulose and chitin, both b-1,4-linked linear saccharides composed of glucose (Glc) or N-acetylglucosamine (GlcNAc), respectively, are the two most abundant biomasses distributed in the plant and animal kingdoms, respectively [1,2]. The biodegradation of these saccharides proceeds via the same path, endoenzymes first degrade higher degree polymerized saccharides into oligosaccharides and then exo-enzymes degrade oligosaccharides into monosaccharides. Cellulase (EC 3.2.1.4) and chitinase (EC 3.2.1.14) are the endo-splitting enzymes, and b-glucosidase (EC 3.2.1.21) and b-N-acetyl-D-hexosaminidase (EC 3.2.1.52) are the exo-splitting enzymes required for cellulose and chitin degradation, respectively [1,2]. The similarity between these two biomolecules hints at a convergent evolution between the degradation enzymes from plants and chitin-containing animals. The recent crystal structural information of exo-splitting enzymes provides evidence of this linkage. Rice (Oryza sativa) b-glucosidase BGlu1 (Os3BGlu7), which exhibits high activity toward cellooligosaccharides [3], belongs to glycosyl hydrolase family 1 according to the CAZy database [4]. The catalysis proceeds via a double displacement mechanism by which two acidic residues act as nucleophile and acid/base catalyst, respectively [5]. The crystal structure of BGlu1 revealed that BGlu1 possesses a classic (b/a)8-barrel catalytic domain constituting a substrate binding pocket with subsites for binding both the leaving Glc (-1 subsite) and the other cellooligosaccharide residues (+1 subsite, +2 subsite and so on) [6,7]. The amino acid residues constituting the subsites for binding the cellooligosaccharide residues, in particular the +1 subsite, are thus determinants for both substrate affinity and specificity. Three residues, Trp358, Ile179 and Tyr131, are found to be crucial for the +1 Glc bindi (...truncated)