The role of cysteine residues in structure and enzyme activity
of a maize beta-glucosidase

J 1999

The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase

ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME et. al.

Basic information

Original name

The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase

Authors

ROTREKL, Vladimír (203 Czech Republic, guarantor), Eliška NEJEDLÁ (203 Czech Republic), Igor KUČERA (203 Czech Republic), Fuad ABDALLAH, Klaus PALME and Břetislav BRZOBOHATÝ (203 Czech Republic)

Edition

European Journal of Biochemistry, 1999, 0014-2956

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

Genetics and molecular biology

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 3.307

RIV identification code

RIV/00216224:14310/99:00001474

Organization unit

Faculty of Science

UT WoS

000084433600040

Keywords in English

beta-glucosidase; cysteine residues; disulfide bridge; structure-function relationships

Abstract

V originále

The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R), or aspartic acid (D) using site-directed mutagenesis and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat. C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino acid resides of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.

Links

IAA5004603, research and development project

Name: Vztah mezi strukturou a funkcí kukuřičné beta-glukozidázy specifické pro konjugáty cytokininů: úloha cysteinových zbytků

Investor: Academy of Sciences of the Czech Republic, Structure-function relationship in maize beta-glucosidase specific for cytokinin glucosides: the role of cysteine residues

VS96096, research and development project

Name: Laboratoř molekulární fyziologie rostlin

Investor: Ministry of Education, Youth and Sports of the CR, Laboratory of Molecular Plant Physiology

Citovat

ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME and Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. European Journal of Biochemistry. 1999, vol. 266, No 3, p. 1056-1065. ISSN 0014-2956.

@article{223837,
   author = {Rotrekl, Vladimír and Nejedlá, Eliška and Kučera, Igor and Abdallah, Fuad and Palme, Klaus and Brzobohatý, Břetislav},
   article_number = {3},
   keywords = {beta-glucosidase; cysteine residues; disulfide bridge; structure-function relationships},
   language = {eng},
   issn = {0014-2956},
   journal = {European Journal of Biochemistry},
   title = {The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase},
   volume = {266},
   year = {1999}
}

TY  - JOUR
ID  - 223837
AU  - Rotrekl, Vladimír - Nejedlá, Eliška - Kučera, Igor - Abdallah, Fuad - Palme, Klaus - Brzobohatý, Břetislav
PY  - 1999
TI  - The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase
JF  - European Journal of Biochemistry
VL  - 266
IS  - 3
SP  - 1056
EP  - 1056
SN  - 00142956
KW  - beta-glucosidase
KW  - cysteine residues
KW  - disulfide bridge
KW  - structure-function relationships
N2  - The maize Zm-p60.1 gene encodes a beta-glucosidase that can release active cytokinins from their storage forms, cytokinin-O-glucosides. Mature catalytically active Zm-p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R), or aspartic acid (D) using site-directed mutagenesis and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea- and glutathione-induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology-based three-dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild-type enzyme, C170 cannot participate in enzyme-substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k'cat. C170 is located on a hydrophobic side of an alpha-helix packed against hydrophobic amino acid resides of beta-strand 4, indicating participation of C170 in stabilization of a (beta/alpha)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k'cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.
ER  -

ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME and Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. \textit{European Journal of Biochemistry}. 1999, vol.~266, No~3, p.~1056-1065. ISSN~0014-2956.

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