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.

Základní údaje

Originální název

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

Autoři

ROTREKL, Vladimír (203 Česká republika, garant), Eliška NEJEDLÁ (203 Česká republika), Igor KUČERA (203 Česká republika), Fuad ABDALLAH, Klaus PALME a Břetislav BRZOBOHATÝ (203 Česká republika)

Vydání

European Journal of Biochemistry, 1999, 0014-2956

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

Genetika a molekulární biologie

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Impakt faktor

Impact factor: 3.307

Kód RIV

RIV/00216224:14310/99:00001474

Organizační jednotka

Přírodovědecká fakulta

UT WoS

000084433600040

Klíčová slova anglicky

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

Štítky

beta-glucosidase, cysteine residues, Disulfide bridge, structure-function relationships

Změněno: 26. 3. 2010 14:02, RNDr. Eliška Nejedlá, CSc.

Anotace

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.

Návaznosti

IAA5004603, projekt VaV

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

Investor: Akademie věd ČR, Vztah mezi strukturou a funkcí kukuřičné beta-glukozidázy specifické pro konjugáty cytokininů: úloha cysteinových zbytků

VS96096, projekt VaV

Název: Laboratoř molekulární fyziologie rostlin

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Laboratoř molekulární fyziologie rostlin

Citovat

ROTREKL, Vladimír, Eliška NEJEDLÁ, Igor KUČERA, Fuad ABDALLAH, Klaus PALME a Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. European Journal of Biochemistry. 1999, roč. 266, č. 3, s. 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 a Břetislav BRZOBOHATÝ. The role of cysteine residues in structure and enzyme activity of a maize beta-glucosidase. \textit{European Journal of Biochemistry}. 1999, roč.~266, č.~3, s.~1056-1065. ISSN~0014-2956.

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