Mechanism of action of α-galactosidase

Mathew, C. Deepal; Balasubramaniam, K.

doi:10.1016/s0031-9422(00)81798-7

Cited by 13 publications

(9 citation statements)

References 13 publications

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“…Hydrophobic cluster analysis of the GGT peptide sequence revealed that the catalytic domain of GGT comprised of an (a/b) 8 -barrel and a C-terminal domain made up of eight b-strands containing a Greek key motif, just like the plant acid a-Gal from rice (Fujimoto et al, 2003). 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.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

2004

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“…regions, are part of an active site [12,22]. An aspartic acid residue might be involved in the active site during the hydrolysis reaction.…”

Section: Comparison Of the Guar C~-galactosidase Amino Acid Sequence mentioning

confidence: 99%

Cloning and nucleotide sequence of the α-galactosidase cDNA from Cyamopsis tetragonoloba (guar)

Overbeeke

et al. 1989

Plant Mol Biol

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Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the alpha-galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH2 terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH2 terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/alpha factor and killer factor. A comparison of the derived amino acid sequence of this alpha-galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the alpha-galactosidase from Escherichia coli.

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“…The kinetic analysis and chemical modification of :xgalactosidases indicated the presence of essential catalytic groups (carboxyl and imidazole groups) and some mechanisms of catalytic action were proposed.b. 7 ) For better understanding of the structure-function relationship of :x-galactosidase, it is necessary to clarify the primary structure. Here we describe the cloning and nucleotide sequence of the :x-galactosidase cDNA from M. vinacea.…”

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confidence: 99%