Purification and Partial Characterization of Maize (<i>Zea mays</i> L.) β-Glucosidase

Esen, Asım

doi:10.1104/pp.98.1.174

Cited by 100 publications

(90 citation statements)

References 29 publications

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“…5). The presented results make C479 a likely candidate for an accessible cysteine residue involved in catalytic activity that was proposed in the rat kidney b-glucosidase [9] and maize b-glucosidase [11] from studies of inhibitors binding covalently to thiol groups. However, definite conclusions on the role of C479 in the Zm-p60.1 catalytic activity must await a detailed analysis of the active centre, which is work we have initiated recently.…”

Section: Discussionmentioning

confidence: 93%

“…Thus, a negative charge in position 479 is not compatible with the enzyme catalytic activity. Interestingly, Esen has proposed the presence of a negatively charged residue near a thiol group suggested to be involved in an enzyme activity of a maize b-glucosidase [11]. Thus, if C479 corresponds to the same cysteine residue, the dramatic effect of the C479D mutation on enzyme activity might be partly caused by repulsions between two adjacent negative charges.…”

Section: Discussionmentioning

confidence: 99%

“…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. Near total activity loss in the presence of 2 m 2-mercaptoethanol indicates that disulfide bond(s) play an important role in maintaining the catalytically active quaternary structure of the enzyme [11].We have cloned a cDNA, Zm-p60.1, coding for a novel maize cytokinin-glucoside-specific b-glucosidase. The mature catalytically active form of Zm-p60.1 is a homodimer located in plastids and chloroplasts.…”

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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: 93%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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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“…Due to recovery of enzyme activity with 2-mercapoethanol after HgCl 2 inhibition, a probable formation of an S-S bridge resulting in homo-dimerization was taken into consideration. However, high amounts of 2-mercapoethanol, which would lead to the dissociation of such a dimer into both subunits [23], did not affect the activity of PNAE. The dimerization obviously does not depend on cysteine residues, but most probably takes place via a contact area of apolar residues [12], as the sequence comparison between the HbHNL and PNAE gives a pronounced homology of 75% (Fig.…”

Section: Discussionmentioning

confidence: 99%

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Mattern-Dogru¹,

Ma²,

Hartmann³

et al. 2002

European Journal of Biochemistry

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In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the a/b hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By sitedirected mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the a/b hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the a/b hydrolase enzyme superfamily.Keywords: polyneuridine aldehyde esterase; active-site residues; site-directed mutagenesis; modelling; a/b hydrolase enzyme superfamily.Polyneuridine aldehyde esterase (PNAE, EC 3.1.1) is a central enzyme in a 10-step biosynthetic pathway expressed in the medicinal plant Rauvolfia serpentina Benth. ex Kurz. The pathway delivers the antiarrhythmic monoterpenoid indole alkaloid ajmaline [1,2]. From the enzymatic point of view, this is presently one of the most detailed investigated examples of a pathway leading to a natural product. Reactions of this complex biosynthetic sequence are catalyzed by membrane-bound and soluble enzymes such as cytochrome P450-dependent hydroxylases, a synthase, several reductases, a methyltransferase and a set of hydrolases including a b-glucosidase, an acetylesterase and the herein described methylesterase PNAE [3]. These enzymes exhibit a high degree of substrate specificity, PNAE being the most specific. PNAE is located in the middle of the biosynthetic chain starting from tryptamine and secologanin and catalyses the conversion of polyneuridine aldehyde into the next stable pathway intermediate, epivellosimine (Fig. 1). The enzyme has been detected in and partially characterized from cell suspension cultures of R. serpentina [4,5]. PNAE has also been highly enriched from cultured plant cells and the cDNA was recently functionally expressed in Escherichia coli [6]. Sequence alignment studies showed highest homologies to an esterase involved in pathogen defence in rice [7] that hydrolyses naphthol esters, and to hydroxynitrile lyases (HNLs) from Hevea brasiliensis [8] and Manihot esculenta [9]. The alignment suggested that PNAE is a new member of the a/b hydrolase superfamily that contains the putative catalytic triad serine, aspartic acid and histidine [10].For the present communication, a more detailed characterization of PNAE was performed by testing the influence of various inhibitors, HNL substrates and by structure elucid...

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“…as these enzymes are apparently localized to the chloroplast (Esen, 1992) and an influence or lack thereof on endogenous cytokinin metabolism has not yet been demonstrated. These enzymes might serve, however, as an example for the low substrate specificity that can characterize some 13-glucosidases (see also Esen, 1993).…”

Section: Analysis Of Rolc/ipt Hybrid Plantsmentioning

confidence: 99%

Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta

et al. 1996

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SummaryThe rolC gene of Agrobacterium rhizogenes T-DNA plays an essential role in the establishment of hairy root disease and its overexpression in transgenic plants causes pleiotropic developmental alterations. This study investigated whether the biological activity of the rolC 13-glucosidase is due to an alteration of the cytokinin balance in planta.HPLC radiocounting assays of [3H]-Iabeled cytokinin glucosides fed exogenously to tobacco leaf disks, to rolC expressing Escherichia coil cells or cell-free extracts showed that cytokinin N3-and O-glucosides are the preferred substrate of the rolC protein. Hydrolysis of N7-and N9-glucosides was not detected at substrate concentrations close to physiological levels. Furthermore, these conjugates were also not active as cytokinins in biotests when fed to rolC-expressing tissues. For analysis of the rolC activity on endogenous cytokinin conjugates the gene was expressed under the transcriptional control of a modified tetracycline-inducible 35S promoter. This was done to avoid possible interference with secondary effects or plant homeostatic mechanisms which could mask primary in planta events when transgenes are expressed constitutively. No changes in the endogenous pool of different cytokinin glucosides, as determined by a newly developed electrospray tandem mass spectroscopy directly coupled to high performance liquid chromatography, were found following chemical induction of the rolC gene. Also the levels of free cytokinins remained unchanged after gene induction. Hybrid tobacco plants expressing the cytokinin-

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Purification and Partial Characterization of Maize (Zea mays L.) β-Glucosidase

Cited by 100 publications

References 29 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

Potential active‐site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site‐directed mutagenesis

Chemically induced expression of the rolC‐encoded β‐glucosidase in transgenic tobacco plants and analysis of cytokinin metabolism: rolC does not hydrolyze endogenous cytokinin glucosides in planta

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