The synthesis, testing and use of 5-fluoro-α-d-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile

Ly, Hoa D.; Howard, Steven; Shum, Kelly; He, Shouming; Zhu, Alex; Withers, Stephen G.

doi:10.1016/s0008-6215(00)00214-7

Cited by 50 publications

(32 citation statements)

References 27 publications

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“…The active site of family 27 acid a-Gals was predicted to contain two carboxyl groups (Mathew and Balasubramaniam, 1987), one serving as the catalytic nucleophile and the other one as the general acid/base catalyst. Recently, crystallization of a rice a-Gal confirmed the identity of Asp in the conserved motif LKYDNC after the fourth b-sheet as the catalytic nucleophile as shown previously for green coffee bean a-Gal (Ly et al, 2000;Fujimoto et al, 2002Fujimoto et al, , 2003. Further, Asp in the motif DIXD was shown to be the acid/base catalyst, and Trp in the conserved motif TPPMGWNSWN is discussed to function in hydrophobic substrate binding of the glycosyl oligosaccharids (Fujimoto et al, 2003).…”

Section: Discussionsupporting

confidence: 64%

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

2004

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Galactan:galactan galactosyltransferase (GGT) is a unique enzyme of the raffinose family oligosaccharide (RFO) biosynthetic pathway. It catalyzes the chain elongation of RFOs without using galactinol (a-galactosyl-myoinositol) by simply transferring a terminal a-galactosyl residue from one RFO molecule to another one. Here, we report the cloning and functional expression of a cDNA encoding GGT from leaves of the common bugle (Ajuga reptans), a winter-hardy long-chain RFO-storing Lamiaceae. The cDNA comprises an open reading frame of 1215 bp. Expression in tobacco (Nicotiana plumbaginifolia) protoplasts resulted in a functional recombinant protein, which showed GGT activity like the previously described purified, native GGT enzyme. At the amino acid level, GGT shows high homologies ([60%) to acid plant a-galactosidases of the family 27 of glycosylhydrolases. It is clearly distinct from the family 36 of glycosylhydrolases, which harbor galactinol-dependent raffinose and stachyose synthases as well as alkaline a-galactosidases. Physiological studies on the role of GGT confirmed that GGT plays a key role in RFO chain elongation and carbon storage. When excised leaves were exposed to chilling temperatures, levels of GGT transcripts, enzyme activities, and long-chain RFO concentrations increased concomitantly. On a whole-plant level, chilling temperatures induced GGT expression mainly in the roots and fully developed leaves, both known RFO storage organs of the common bugle, indicating an adaptation of the metabolism from active growth to transient storage in the cold.

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Section: Discussionsupporting

confidence: 64%

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

2004

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“…This method requires specifically designed enzyme inhibitor molecules that create a stable bond between the enzyme and inhibitor. Using this strategy, the nucleophilic aspartic acid of the GH27 ␣-Gals from Phanerochaete chrysosporium and Coffea arabica (Uniprot, Q42656) was identified (22,35).…”

Section: Vol 188 2006mentioning

confidence: 99%

Identification of a Novel α-Galactosidase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

Brouns

Smits

et al. 2006

J Bacteriol

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Sulfolobus solfataricus is an aerobic crenarchaeon that thrives in acidic volcanic pools. In this study, we have purified and characterized a thermostable ␣-galactosidase from cell extracts of S. solfataricus P2 grown on the trisaccharide raffinose. The enzyme, designated GalS, is highly specific for ␣-linked galactosides, which are optimally hydrolyzed at pH 5 and 90°C. The protein consists of 74.7-kDa subunits and has been identified as the gene product of open reading frame Sso3127. Its primary sequence is most related to plant enzymes of glycoside hydrolase family 36, which are involved in the synthesis and degradation of raffinose and stachyose. Both the galS gene from S. solfataricus P2 and an orthologous gene from Sulfolobus tokodaii have been cloned and functionally expressed in Escherichia coli, and their activity was confirmed. At present, these Sulfolobus enzymes not only constitute a distinct type of thermostable ␣-galactosidases within glycoside hydrolase clan D but also represent the first members from the Archaea.␣-Galactosidases (␣-Gals) (EC 3.2.1.22) are a widespread class of enzymes that liberate galactose from the nonreducing end of sugars. In bacteria, yeasts, and fungi, these enzymes are usually involved in the degradation of various plant saccharides, which can then serve as a carbon and energy source for growth. Plants synthesize ␣-galactosides such as raffinose and stachyose as the major energy storage molecules in leaves, roots, and tubers. Moreover, these oligosaccharides have been associated with cold and desiccation tolerance of seeds (28). Both raffinose and stachyose are produced by two specialized synthases (raffinose synthase [EC 2.4.1.82] and stachyose synthase [EC 2.4.1.67]) that use galactinol as a galactosyl donor (39). The oligosaccharides are degraded during seed germination by the action of two distinct types of ␣-Gals that differ in their optimal pH of catalysis. While the acid ␣-Gal type is most likely active in the acidic environment of the vacuole and the apoplasm, the alkaline ␣-Gal type probably catalyzes galactose release in the more neutral or alkaline cytoplasm (4,17,31). Mammals express an ␣-Gal and an ␣-N-acetylgalactosaminidase (␣-NAGal) in lysosomal bodies to degrade glycolipids, glycoproteins, and oligosaccharides. In humans, mutations in

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“…␣-Gals from eukaryotes have high amino acid sequence similarities and are basically classified into family 27, whereas bacterial ␣-Gals are grouped into families 4 and 36. The hydrolysis mechanism of ␣-Gal is known to proceed with retention of the stereochemistry at the anomeric center through the double displacement mechanism, and the nucleophile of the catalytic residue is an aspartic acid that is highly conserved in all known family 27 ␣-Gals and other glycosyl hydrolases (21,22). Recently, the crystal structure of chicken ␣-N-acetylgalactosaminidase (␣-NAGal; E.C.…”

Section: ␣-Galactosidases (␣-Galsmentioning

confidence: 99%

Crystal Structure of Rice α-Galactosidase Complexed with D-Galactose

Fujimoto

Kaneko

Momma

et al. 2003

Journal of Biological Chemistry

107

131

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␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. The crystal structure of rice ␣-galactosidase has been determined at 1.5Å resolution using the multiple isomorphous replacement method. The structure consisted of a catalytic domain and a C-terminal domain and was essentially the same as that of ␣-N-acetylgalactosaminidase, which is the same member of glycosyl hydrolase family 27. The catalytic domain had a (␤/␣) 8 -barrel structure, and the C-terminal domain was made up of eight ␤-strands containing a Greek key motif. The structure was solved as a complex with D-galactose, providing a mode of substrate binding in detail. The D-galactose molecule was found bound in the active site pocket on the C-terminal side of the central ␤-barrel of the catalytic domain. The D-galactose molecule consisted of a mixture of two anomers present in a ratio equal to their natural abundance. Structural comparisons of rice ␣-galactosidase with chicken ␣-N-acetylgalactosaminidase provided further understanding of the substrate recognition mechanism in these enzymes. ␣-Galactosidases (␣-Gals1 ; E.C. 3.2.1.22) catalyze the hydrolysis of ␣-1,6-linked galactosyl residues from galacto-oligosaccharides and polymeric galacto-(gluco)mannans. ␣-Gals are widely distributed in animals, plants, and microorganisms.In humans, ␣-Gal is a lysosomal exoglycosidase that cleaves the terminal ␣-galactose residue from glycolipids and glycoproteins. Mutations in the ␣-Gal gene cause incomplete degradation of carbohydrates, resulting in Fabry disease (1, 2). In plants, galactomannan is one of the major storage polysaccharides in seeds, and ␣-Gal is one of the key enzymes in the degradation of cell wall galactomannan during germination (3, 4). Raffinose and stachyose in beans are known to cause flatulence, and ␣-Gal has the potential to alleviate these symptoms (5). In the sugar beet industry, ␣-Gal has been used to increase the sucrose yield by eliminating raffinose, which prevents normal crystallization of beet sugar (6).We have purified and sequenced several ␣-Gals from Mortierella vinacea, Penicillium purpurogenum, Thermus sp. T2, and Oryza sativa L. and have elucidated the substrate specificities of these enzymes using two types of the galactomannooligosaccharides, 63 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose and 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose (7-13). The results showed that ␣-Gals have a diverse preference for substrates. The M. vinacea ␣-Gal I and yeast ␣-Gals were specific only for 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotriose, which has an ␣-galactosyl residue (designated the terminal ␣-galactosyl residue) linked to the non-reducing end mannose of ␤-1,4-mannotriose (14, 15). Aspergillis niger ␣-Gal and P. purpurogenum ␣-Gal, however, showed a preference for 6 3 -mono-␣-D-galactopyranosyl-␤-1,4-mannotetraose, which has an ␣-galactosyl residue (designated the side-chain ␣-galactosyl residue) attached to the inner mannose of ␤-1,4-ma...

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The synthesis, testing and use of 5-fluoro-α-d-galactosyl fluoride to trap an intermediate on green coffee bean α-galactosidase and identify the catalytic nucleophile

Cited by 50 publications

References 27 publications

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

Cloning, Functional Expression, and Characterization of the Raffinose Oligosaccharide Chain Elongation Enzyme, Galactan:Galactan Galactosyltransferase, from Common Bugle Leaves

Identification of a Novel α-Galactosidase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

Crystal Structure of Rice α-Galactosidase Complexed with D-Galactose

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