The crystal structure of a cyanogenic β-glucosidase from white clover, a family 1 glycosyl hydrolase

Barrett, Tracey E.; Suresh, C.G.; Tolley, SP; Dodson, E.J.; Ma, Huiqin

doi:10.1016/s0969-2126(01)00229-5

Cited by 201 publications

(186 citation statements)

References 40 publications

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“…The location of C170 in the model precludes participation of this cysteine residue in formation of a disulfide bridge in Zm-p60.1. Furthermore, an intramolecular disulfide bridge, formed between cysteine residues in the positions equivalent to C205 and C211, was identified in the crystal structure of CBG [7]. Finally, in plant b-glucosidases and b-thiolglucosidases with highly related amino-acid sequences, and acting as dimers, a cysteine residue in the position equivalent to the Zm-p60.1 position 205 is conserved followed by a second cysteine residue separated by 5±9 amino-acid residues (C211 in the Zm-p60.1; Fig.…”

Section: Discussionmentioning

confidence: 98%

“…7; only monomer unit of Zm-p60.1 dimer is shown for simplicity) was constructed on the basis of the X-ray analysis of a closely related cyanogenic b-glucosidase CBG [7]. In the CBG structure, there are two cysteine residues forming a disulfide bridge in positions equivalent to C205 and C211.…”

Section: Homology Modelingmentioning

confidence: 99%

“…The threedimensional structure of a cyanogenic b-glucosidase from white clover (CBG) has been elucidated recently [7]. The overall fold of the CBG molecule is a (b/a) 8 barrel.…”

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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: 98%

Section: Homology Modelingmentioning

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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“…Three-dimensional structures are available for a large number of Family1 enzyme, the first solved being that of the white clover (Trifolium repens) cyanogenic β-glucosidase [14]. As members of Clan GH-A they have a classical (α/β) 8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [15,16].…”

Section: Three-dimensional Structures Of Gh1mentioning

confidence: 99%

Extremophilic Carbohydrate Active Enzymes (CAZymes)

Lee¹,

Park²,

Park³

2017

JNHFE

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Carbohydrate active enzymes (CAZymes) are a large class of enzymes, which build and breakdown the complex carbohydrates of the cell. On the basis of their amino acid sequences they are classified in families that show conserved catalytic mechanism, structure, and active site residues, but may conflict each other in substrate specificity. The CAZymes provides a continuously updated list of the glycoside hydrolase families, GHs. This group of enzymes is classified based on functional similarity, but today they are classified into 108 GHs on the basis of amino acid sequence similarity. Despite their similarities to enzymes with known functions, their primary functions are still unclear. Based on these criteria, β-galactosidase activities are now divided into four different families: GH1, GH2, GH35 and GH42, among which the better studied GH2 includes β-galactosidase from Escherichia coli, Aspergillus, Bacillus megatherium, and Sulfolobus solfataricus, while those from thermophilic, psychrophilic and halophilic organisms belong to GH42. Lactase is often confused as an alternate name for β-galactosidase, but it is actually simply a subclass (small subunit) of β-galactosidase.(β-D-galactoside galactohydrolase, EC 3.2.1.23) that catalyses hydrolysis of the galactosyl moiety from non-reducing termini of oligosaccharides or from glycosides. Most genes encoding GH42 enzymes are from prokaryotes that are unable to grow on lactose as a sole carbon source and at least two GH42 β-galactosidase do not cleave lactose in vitro. The determination of growth on lactose can be complicated by the multiple β-galactosidases because not all of the β-galactosidase is acting as lactases in vitro. Β-Galactosidase hydrolyses the β-1, 4-D-galactosidic linkage of lactose, as well as those of related chromogens, o-nitrophenyl-β-dgalactopyranoside (ONPG), p-nitrophenyl-β-d-galactopyranoside (PNPG) and 6-bromo-2-naphthyl-galactopyranoside (BNG). This enzyme has been purified and characterized from various sources, including plants, animals, and many microorganisms. In humans, lactase is present predominantly along the brush border membrane of the differentiated enterocytes lining the villi of the small intestine. Lactase is essential for digestive hydrolysis of lactose in milk. Deficiency of the enzyme causes lactose intolerance. Several 3D structures are available for the GH1 and GH2 β-galactosidase for which the catalytic residues have experimentally been determined, while three 3D structures of β-galactosidase from E. coli, Penicillium sp and Thermus thermophilus A4 are available for both the GH35 and GH42 families. Glycoside hydrolasesGlycoside hydrolases are enzymes that catalyze the hydrolysis of the glycosidic linkage of glycosides, leading to the formation of a sugar hemiacetal or hemiketal and the corresponding free aglycon. Glycoside hydrolases are also referred to as glycosidases, and sometimes also as glycosyl hydrolases. Glycoside hydrolases can catalyze the hydrolysis of O-, N-and S-linked glycosides ( Figure 1). Endo/Exo:Exo / ...

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“…The NEP sequence motif, of which the Glu residue is an acid/ base catalyst, was found at residues 257-259, and the sequence ITENG, of which the Glu residue is a catalytic nucleophile of β-glucosidases, was also found at residues 468-472 (Jenkins et al 1995;Keresztessy et al 1994). The residues involved in the binding of the glycone (glucose) Copyright © 2015 The Japanese Society for Plant Cell and Molecular Biology moiety are highly conserved in all GH1s (Barrett et al 1995;Rye and Withers 2000;Saino et al 2014;Sue et al 2006;Zechel and Withers 2000), and these residues were also found at Gln-108, His-212, Asn-257, Glu-258, Glu-526, and Trp-527 in the DeF26G1 sequence ( Figure 5). …”

Section: Isolation Of Full-length Def26g1 Cdnamentioning

confidence: 99%

Identification of furostanol glycoside 26-<i>O</i>-β-glucosidase involved in steroidal saponin biosynthesis from <i>Dioscorea esculenta</i>

Nakayasu

Kawasaki

Lee

et al. 2015

Plant Biotechnology

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Steroidal saponins are natural surfactants with various biological activities, and the tubers of Dioscorea, known as yam, contain a furostanol glycoside protodioscin and a spirostanol glycoside dioscin, which are valuable saponins required for semi-synthetic production of pharmaceutical steroidal drugs. Steroidal saponins are biosynthesized from cholesterol via several steps of oxygenation and transglycosylation, and a β-glucosidase is involved in the hydrolytic conversion from furostanol glycosides to spirostanol glycosides. To investigate steroidal saponin biosynthesis in Dioscorea spps, comparative transcriptome analysis of high saponin producers, D. esculenta and D. cayenensis, and a low producer, D. alata, was performed using 454 pyrosequencing. In this study, we isolated and characterized a β-glucosidase (DeF26G1) from D. esculenta. The DeF26G1 cDNA encodes a family 1 glucosidase, and the DeF26G1 transcript was present at high levels in D. esculenta but not detected in D. alata. The recombinant DeF26G1 protein hydrolyzed the 26-O-glycosidic bond of protodioscin to form dioscin, indicating that the DeF26G1 gene encodes furostanol glycoside 26-O-β-glucosidase. These results suggested that DeF26G1 is involved in the conversion of furostanol saponins to spirostanol saponins, which seems to be related to biological defense response in the leaves of Dioscorea plants.

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The crystal structure of a cyanogenic β-glucosidase from white clover, a family 1 glycosyl hydrolase

Cited by 201 publications

References 40 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

Extremophilic Carbohydrate Active Enzymes (CAZymes)

Identification of furostanol glycoside 26-<i>O</i>-β-glucosidase involved in steroidal saponin biosynthesis from <i>Dioscorea esculenta</i>

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