Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis

0440809 - BFÚ 2015 RIV GB eng J - Journal Article
Filipi, T. - Mazura, P. - Janda, L. - Kiran, N.S. - Brzobohatý, Břetislav
Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis.
Phytochemistry. Roč. 74, FEB2012 (2012), s. 40-48. ISSN 0031-9422. E-ISSN 1873-3700
Institutional support: RVO:68081707
Keywords : beta-Glucosidase * cis-Zeatin-O-beta-D-glucopyranoside * Cytokinin metabolism
Subject RIV: BO - Biophysics
Impact factor: 3.050, year: 2012

The maize beta-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-beta-D-glucopyranoside versus the trans-zeatin-O-beta-D-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer.
Permanent Link: http://hdl.handle.net/11104/0243959