Expression of a Zeatin-O-Glucoside-Degrading [beta]-Glucosidase in Brassica napus

Falk, Anders; Rask, Lars

doi:10.1104/pp.108.4.1369

Cited by 88 publications

(48 citation statements)

References 24 publications

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“…The maize b-glucosidase dimer has been reported to dissociate into monomers upon 2-mercaptoethanol treatment [1]. The B. napus b-glucosidase dimer has also been shown to dissociate into monomers upon reduction and alkylation [10], leading the authors conclude that the protein subunits are covalently linked by a disulfide bond. However (His) 6 Zm-p60.r dimer dissociation into monomers can be achieved also by a simple urea treatment without the need for any reducing agent (Fig.…”

Section: Discussionmentioning

confidence: 76%

“…In rat kidney b-glucosidase, at least one functionally essential and accessible thiol group was detected using inhibitors binding covalently to thiol groups [9]. A critical role in maintaining dimer structure has been assigned to a disulfide bridge in Brassica napus b-glucosidase [10]. In a maize b-glucosidase, the loss of enzyme activity upon treatment with alkylating agents and the reversible inhibition of the enzyme by Hg 2+ and Ag + , which can be completely overcome by adding 2-mercaptoethanol in molar excess (up to 30 mm) over the inhibitory metal ion, suggests that the active centre of the enzyme includes a cysteine that is required for activity.…”

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confidence: 99%

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The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Rotrekl¹,

Nejedlá²,

Kučera³

et al. 1999

European Journal of Biochemistry

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The maize Zm-p60.1 gene encodes a b-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 K m 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 H cat . C170 is located on a hydrophobic side of an a-helix packed against hydrophobic aminoacid residues of b-strand 4, indicating participation of C170 in stabilization of a (b/a) 8 barrel structure in the enzyme. In C479A/D/R/S mutants, K m and k H cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.Keywords: b-glucosidase; cysteine residues; disulfide bridge; structure±fuction relationships.Current research on b-glucosidases of animals, plants and microorganisms has significant scientific, clinical and economic implications (reviewed in [1]). In plants, b-glucosidases have been implicated in various aspects of growth, productivity and defence, and reactions such as cyanogenesis related to food and feed toxicity. In addition, recent data indicate that plant b-glucosidases may be involved in the metabolism of plant hormones, whose storage forms occur as b-glucosides and are activated by b-glucosidases [2,3]. Thus, information on structure±function relationships in the b-glucosidases is of obvious importance for understanding their biological functions, and engineering their catalytic and molecular properties for industrial and field applications.b-Glucosidases catalyse the hydrolysis of aryl and alkyl-b-dglucosides as well as glucosides with only carbohydrate moieties (such as cellobiose). Catalysis involves two essential carboxylates, one acting as a proton donor and the other as a nucleophile [4]. Cleavage of the glucosidic bond results in retention of the configuration at the anomeric carbon atom.Plant b-glucosidases are classified into family 1 of glycos...

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Section: Discussionmentioning

confidence: 76%

mentioning

confidence: 99%

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Rotrekl¹,

Nejedlá²,

Kučera³

et al. 1999

European Journal of Biochemistry

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“…The recombinant enzyme cleaves cytokinin O-glucosides as well as kinetin-N3-glucoside, indicating low substrate specificity (Brzobohatý et al, 1993). A similar gene was isolated from Brassica napus (Falk and Rask, 1995). The recombinant enzyme converted zeatin-O-glucoside to zeatin; however, other substrates, such as related Oglucosides and cytokinin N-glucosides, were not tested.…”

Section: Cytokinin Metabolismmentioning

confidence: 99%

Cytokinins: Biosynthesis metabolism and perception

Mok

Martin

Mok

2000

In Vitro Cell.Dev.Biol.-Plant

108

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SummaryCytokinins are essential hormones for plant growth and development. They are also of vital importance for in vitro manipulations of plant cells and tissues. The biological activities and chemistry of cytokinins are well defined but very little is known about their mode of action and it is only recently that cytokinin genes have been identified in plants. This review summarizes the current status of knowledge on cytokinin biosynthesis, metabolism and signal transduction, with an emphasis on genes encoding metabolic enzymes and putative receptors, and genes rapidly induced by cytokinins.

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“…The sense and the anti-sense probes corresponding to base pairs 277-524 in the iMyAP9 cDNA clone and to base pairs 1552-1789 of the vegetatively expressed MYRI cDNA-clone (Falk et al, 1992) were labeled as previously described (Falk and Rask, 1995). Plant tissues were treated as described by Falk and Rask (1995), except that the leaves were fixed by vacuum infiltration in a SpeedVac Concentrator SVC 100H (Savant Instruments). The final wash was in 0.2XNaCKit at 55OC for 1 h.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Wound‐Induced Myrosinase‐Associated Protein Transcript in Brassica Napus Plants

Taipalensuu

Andréasson

Eriksson

et al. 1997

European Journal of Biochemistry

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Two slightly differing cDNA clones corresponding to the wound-inducible form of a previously characterized seed myrosinase-associated protein (MyAP) have been isolated from Brassica nupus L. The transcripts corresponding to the induced MyAP (iMyAP) were found to be developmentally regulated in various vegetative organs. Both young and old leaves exhibited wound-inducible iMyAP expression. Furthermore, in the young plant, wounding resulted in a systemic increase in leaves located both acropetally and basipetally to the wounded leaf. Also, the iMyAP transcripts were induced by methyl jasmonate, jasmonic acid and abscisic acid. The different inductions could be antagonized by salicylic acid. A general responsiveness in methyl-jasmonate-treated leaves was demonstrated by in situ hybridization. No effect on the amount of iMyAP transcript was detected after feeding the plants with the ethylene precursor 1-aminocyclopropane-1 -carboxylic acid. The similarity between MyAP and a lipase from Arubidopsis thuliana indicated a possible function in liberating acylated glucosinolates from their acyl group, thereby making them available for hydrolysis by the myrosinase enzymes. We also report on a reduction in the amount of myrosinase transcripts derived from the vegetatively expressed MB-gene family after treatment with exogenously applied salicylic acid or abscisic acid.

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Expression of a Zeatin-O-Glucoside-Degrading [beta]-Glucosidase in Brassica napus

Cited by 88 publications

References 24 publications

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase

Cytokinins: Biosynthesis metabolism and perception

Regulation of the Wound‐Induced Myrosinase‐Associated Protein Transcript in Brassica Napus Plants

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