title: Enhancement of catalysis and functional expression of a bacterial laccase by single amino acid replacement.
abstract: Structure-function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp. HR03 with the ability of bilirubin oxidation besides its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp(500) located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase kcat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveal the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Nikoo
authors.initials: N
authors.lastname: Nasoohi
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Mahdi
authors.initials: M
authors.lastname: Mohammadian
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001663:Bilirubin / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D042845:Laccase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D042845:Laccase; D001663:Bilirubin
keywords: 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); ABTS; CD; Catalytic efficiency; Functional expression; IPTG; Laccase; PMSF; SGZ; Site-directed mutagenesis; T1 copper; T1 copper-binding loop; circular dichroism; isopropyl-β-d-1-thiogalactopyranoside; phenylmethylsulfonyl fluoride; syringaldazine; type-1 copper