title: Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis γ-glutamyltranspeptidase.
abstract: Bacillus licheniformis γ-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes. Here we investigated the role of a conserved Asn450 residue in BlGGT through site-directed mutagenesis and molecular characterization of four relevant variants. Substitution of Asn450 by arginine resulted in a significant reduction in the catalytic activity of BlGGT. Conversely, N450A and N450D displayed an enhanced activity. The catalytic efficiency of BlGGT was calculated to be 16.04mM(-1)s(-1), but this value was either decreased to 8.93mM(-1)s(-1) in N450K or increased to more than 123.65mM(-1)s(-1) in N450A and N450D. In addition, the ratio of transpeptidation to hydrolysis was increased from 3.5 to more than 7.6 by the mutations. Structural analyses showed that fluorescence, circular dichroism spectra and thermal denaturation profiles of mutant proteins were essentially consistent with those of BlGGT. However, guanidine hydrochloride (GdnHCl)-induced transition was significantly reduced in comparison with the wild-type enzyme. Molecular modeling suggests that residue Asn450 of BlGGT is important to create suitable environments for both autoprocessing and catalytic reactions.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nangang District, Taipei City 11529, Taiwan.
authors.forename: Min-Guan
authors.initials: MG
authors.lastname: Lin
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Meng-Chun
authors.initials: MC
authors.lastname: Chi
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
authors.forename: Yu-Yi
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Department of Chemistry, National Cheng Kung University, Tainan City 701, Taiwan.
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.affiliation: Department of Food Science and Technology, Hungkuang University, 1018 Taiwan Boulevard, Shalu District, Taichung City 43302, Taiwan. Electronic address: hflo@sunrise.hk.edu.tw.
authors.forename: Hui-Fen
authors.initials: HF
authors.lastname: Lo
authors.affiliation: Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan. Electronic address: llin@mail.ncyu.edu.tw.
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
mesh_terms: D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry / Q000235:genetics; D000069976:Bacillus licheniformis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D001216:Asparagine; D005723:gamma-Glutamyltransferase
keywords: Asparagine; Site-directed mutagenesis; γ-Glutamyltranspeptidase