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

J 2012

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

FILIPI, Tomáš, Pavel MAZURA, Lubomír JANDA, Nagavalli Subbanna KIRAN, Břetislav BRZOBOHATÝ et. al.

Basic information

Original name

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

Authors

FILIPI, Tomáš, Pavel MAZURA, Lubomír JANDA, Nagavalli Subbanna KIRAN and Břetislav BRZOBOHATÝ

Edition

Phytochemistry, Oxford, UK, Elsevier Science, 2012, 0031-9422

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 3.050

Organization unit

Central European Institute of Technology

DOI

http://dx.doi.org/10.1016/j.phytochem.2011.10.008

UT WoS

000300815700004

Keywords in English

(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

Abstract

V originále

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.

Links

ED1.1.00/02.0068, research and development project

Name: CEITEC - central european institute of technology

LC06034, research and development project

Name: Regulace morfogeneze rostlinných buněk a orgánů

Investor: Ministry of Education, Youth and Sports of the CR, Regulation of morphogenesis of plant cells and organs

Citovat

FILIPI, Tomáš, Pavel MAZURA, Lubomír JANDA, Nagavalli Subbanna KIRAN and Břetislav BRZOBOHATÝ. Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis. Phytochemistry. Oxford, UK: Elsevier Science, 2012, vol. 74, p. 40-48. ISSN 0031-9422. Available from: https://dx.doi.org/10.1016/j.phytochem.2011.10.008.

@article{968261,
   author = {Filipi, Tomáš and Mazura, Pavel and Janda, Lubomír and Kiran, Nagavalli Subbanna and Brzobohatý, Břetislav},
   article_location = {Oxford, UK},
   doi = {http://dx.doi.org/10.1016/j.phytochem.2011.10.008},
   keywords = {(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},
   language = {eng},
   issn = {0031-9422},
   journal = {Phytochemistry},
   title = {Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis},
   volume = {74},
   year = {2012}
}

TY  - JOUR
ID  - 968261
AU  - Filipi, Tomáš - Mazura, Pavel - Janda, Lubomír - Kiran, Nagavalli Subbanna - Brzobohatý, Břetislav
PY  - 2012
TI  - Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis
JF  - Phytochemistry
VL  - 74
SP  - 40-48
EP  - 40-48
PB  - Elsevier Science
SN  - 00319422
KW  - (alpha/beta)(8) Barrel
KW  - beta-Glucosidase
KW  - cis-Zeatin-O-beta-D-glucopyranoside
KW  - Cytokinin metabolism
KW  - Glycosidase
KW  - Protein engineering
KW  - Site-directed random mutagenesis
KW  - Substrate specificity
KW  - trans-Zeatin-O-beta-D-glucopyranoside
KW  - trans-Zeatin-9-beta-D-glucopyranoside
N2  - 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.
ER  -

FILIPI, Tomáš, Pavel MAZURA, Lubomír JANDA, Nagavalli Subbanna KIRAN and Břetislav BRZOBOHATÝ. Engineering the cytokinin-glucoside specificity of the maize beta-D-glucosidase Zm-p60.1 using site-directed random mutagenesis. \textit{Phytochemistry}. Oxford, UK: Elsevier Science, 2012, vol.~74, p.~40-48. ISSN~0031-9422. Available from: https://dx.doi.org/10.1016/j.phytochem.2011.10.008.

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