title: Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis.
abstract: The maize β-D-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-D-glucopyranoside versus the trans-zeatin-O-β-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-D-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.
authors.affiliation: Laboratory of Plant Molecular Biology, Institute of Biophysics AS CR, vvi and Mendel University in Brno, Zemědělská 1, CZ-613 00 Brno, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Filipi
authors.forename: Pavel
authors.initials: P
authors.lastname: Mazura
authors.forename: Lubomír
authors.initials: L
authors.lastname: Janda
authors.forename: Nagavalli S
authors.initials: NS
authors.lastname: Kiran
authors.forename: Břetislav
authors.initials: B
authors.lastname: Brzobohatý
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D001665:Binding Sites; D003583:Cytokinins / Q000378:metabolism*; D017343:Genes, Plant; D005960:Glucosides / Q000378:metabolism*; D006868:Hydrolysis; D007536:Isomerism; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D015026:Zeatin / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D000596:Amino Acids; D003583:Cytokinins; D005960:Glucosides; D010940:Plant Proteins; D015026:Zeatin; D001617:beta-Glucosidase