title: Characterization and identification of essential residues of the glycoside hydrolase family 64 laminaripentaose-producing-β-1, 3-glucanase.
abstract: Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.
authors.affiliation: Department of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan.
authors.forename: Keshab Lal
authors.initials: KL
authors.lastname: Shrestha
authors.forename: Sheng-Wen
authors.initials: SW
authors.lastname: Liu
authors.forename: Chin-Ping
authors.initials: CP
authors.lastname: Huang
authors.forename: Hsin-Mao
authors.initials: HM
authors.lastname: Wu
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Wang
authors.forename: Yaw-Kuen
authors.initials: YK
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D019810:Sodium Azide; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D047071:beta-Glucans; C038459:curdlan; D019810:Sodium Azide; D043326:Glucan 1,3-beta-Glucosidase