title: Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect β-N-acetyl-D-hexosaminidase.
abstract: The chemical similarity of cellulose and chitin supports the idea that their corresponding hydrolytic enzymes would bind β-1,4-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 (Trp(490), Glu(328), Val(327) in OfHex1, and Trp(358), Tyr(131) and Ile(179) in BGlu1) were identified as being conserved in the +1 sugar binding site. OfHex1 Glu(328) together with Trp(490) was confirmed to be necessary for substrate binding. The mutant E328A exhibited a 8-fold increment in K(m) for (GlcNAc)(2) and a 42-fold increment in K(i) for TMG-chitotriomycin. A crystal structure of E328A in complex with TMG-chitotriomycin was resolved at 2.5 Å, revealing the obvious conformational changes of the catalytic residues (Glu(368) and Asp(367)) 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 β-1,2-linked GlcNAc in contrast to the wild-type. Thus, Glu(328) and Val(327) were identified as important for substrate-binding and as glycosidic-bond determinants. A structure-based sequence alignment confirmed the spatial conservation of these three residues in most plant cellulolytic, insect and bacterial chitinolytic enzymes.
authors.affiliation: School of Life Science and Biotechnology, Dalian University of Technology, Dalian, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhou
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.forename: Xu
authors.initials: X
authors.lastname: Shen
authors.forename: Wenqing
authors.initials: W
authors.lastname: Zhang
authors.forename: Jianzhen
authors.initials: J
authors.lastname: Zhang
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism*; D002686:Chitin / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D007915:Lepidoptera / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology; D013402:Sugar Alcohols / Q000378:metabolism; D013780:Tetroses / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D013402:Sugar Alcohols; C528826:TMG-chitotriomycin; D013780:Tetroses; D002686:Chitin; C048468:cellotetraose; D002482:Cellulose; D001617:beta-Glucosidase; D001619:beta-N-Acetylhexosaminidases