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

Article in a journal

Field of Study

10600 1.6 Biological sciences

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

is not subject to a state or trade secret

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

In the original language

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

Cite

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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