title: Substrate binding of a GH5 endoglucanase from the ruminal fungus Piromyces rhizinflata.
abstract: The endoglucanase EglA from Piromyces rhizinflata found in cattle stomach belongs to the GH5 family of glycoside hydrolases. The crystal structure of the catalytic domain of EglA shows the (β/α)(8)-barrel fold typical of GH5 enzymes. Adjacent to the active site of EglA, a loop containing a disulfide bond not found in other similar structures may participate in substrate binding. Because the active site was blocked by the N-terminal His tag of a neighbouring protein molecule in the crystal, enzyme-substrate complexes could not be obtained by soaking but were prepared by cocrystallization. The E154A mutant structure with a cellotriose bound to the -3, -2 and -1 subsites shows an extensive hydrogen-bonding network between the enzyme and the substrate, along with a stacking interaction between Trp44 and the -3 sugar. A possible dimer was observed in the crystal structure, but retention of activity in the E242A mutant suggested that the enzyme probably does not function as a dimer in solution. On the other hand, the first 100 amino acids encoded by the original cDNA fragment are very similar to those in the last third of the (β/α)(8)-barrel fold, indicating that EglA comprises at least two catalytic domains acting in tandem.
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei 10617, Taiwan.
authors.forename: Chih Wen
authors.initials: CW
authors.lastname: Tseng
authors.forename: Tzu Ping
authors.initials: TP
authors.lastname: Ko
authors.forename: Rey Ting
authors.initials: RT
authors.lastname: Guo
authors.forename: Jian Wen
authors.initials: JW
authors.lastname: Huang
authors.forename: Hao Ching
authors.initials: HC
authors.lastname: Wang
authors.forename: Chun Hsiang
authors.initials: CH
authors.lastname: Huang
authors.forename: Ya Shan
authors.initials: YS
authors.lastname: Cheng
authors.forename: Andrew H J
authors.initials: AH
authors.lastname: Wang
authors.forename: Je Ruei
authors.initials: JR
authors.lastname: Liu
mesh_terms: D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D009154:Mutation; D020089:Piromyces / Q000201:enzymology*; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D040681:Structural Homology, Protein; D013379:Substrate Specificity
chemical_list: D002480:Cellulase
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