Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
Jazyk angličtina Země Velká Británie, Anglie Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
22079107
DOI
10.1016/j.phytochem.2011.10.008
PII: S0031-9422(11)00477-8
Knihovny.cz E-zdroje
- MeSH
- aminokyseliny metabolismus MeSH
- beta-glukosidasa chemie genetika MeSH
- cytokininy metabolismus MeSH
- glukosidy metabolismus MeSH
- hydrolýza MeSH
- isomerie MeSH
- kukuřice setá chemie enzymologie genetika MeSH
- molekulární konformace MeSH
- mutace MeSH
- mutageneze cílená metody MeSH
- rostlinné geny MeSH
- rostlinné proteiny chemie genetika MeSH
- sekvence aminokyselin MeSH
- substrátová specifita MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zeatin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- aminokyseliny MeSH
- beta-glukosidasa MeSH
- cytokininy MeSH
- glukosidy MeSH
- rostlinné proteiny MeSH
- zeatin MeSH
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.
Citace poskytuje Crossref.org
Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants
New Insights Into the Metabolism and Role of Cytokinin N-Glucosides in Plants
High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing
An automated method to evaluate the enzyme kinetics of β-glucosidases
Combining rational and random strategies in β-glucosidase Zm-p60.1 protein library construction
