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

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FILIPI Tomáš MAZURA Pavel JANDA Lubomír KIRAN Nagavalli Subbanna BRZOBOHATÝ Břetislav

Druh Článek v odborném periodiku
Časopis / Zdroj Phytochemistry
Fakulta / Pracoviště MU

Lékařská fakulta Přírodovědecká fakulta Centrální řídící struktura projektu CEITEC Středoevropský technologický institut

Citace
Doi http://dx.doi.org/10.1016/j.phytochem.2011.10.008
Obor Biochemie
Klíčová slova (alpha/beta)(8) Barrel; beta-Glucosidase; cis-Zeatin-O-beta-D-glucopyranoside; Cytokinin metabolism; Glycosidase; Protein engineering; Site-directed random mutagenesis; Substrate specificity; trans-Zeatin-O-beta-D-glucopyranoside; trans-Zeatin-9-beta-D-glucopyranoside
Popis 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. 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-beta-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. (C) 2011 Elsevier Ltd. All rights reserved.
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