The Molecular Mechanism of Thermostable α-Galactosidases AgaA and AgaB Explained by X-ray Crystallography and Mutational Studies

Merceron, Romain; Foucault, M.; Haser, R.; Mattes, Ralf; Watzlawick, Hildegard; Gouet, Patrice

doi:10.1074/jbc.m112.394114

Cited by 40 publications

(60 citation statements)

References 43 publications

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“…A further example concerns two homologous α-galactosidases (AgaA and AgaB) from family GH36 (clan GH-D) [162]. Despite being highly related (97 % identity), AgaA displays a relatively low K m value for raffinose (K m = 3.8 mM) and exhibits high hydrolytic activity and no detectable ability to catalyse transglycosylation.…”

Section: Enzyme Engineeringmentioning

confidence: 99%

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Bissaro

Monsan

Fauré

et al. 2015

Biochemical Journal

136

152

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Carbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases.

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Section: Enzyme Engineeringmentioning

confidence: 99%

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Bissaro

Monsan

Fauré

et al. 2015

Biochemical Journal

136

152

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“…Two Asp residues are required for catalysis, which are positioned on opposite sides of the labile glycosidic bond (Fujimoto et al, 2003). RafS, StaS, and alkaline α-galactosidases belong to the related GH36, but no structural information is yet available on plant members within this family (Vanhaecke, 2010), although a few microbial structures became available (Fredslund et al, 2011; Merceron et al, 2012). To our knowledge, no in depth structure–function research has been performed toward donor and acceptor substrate specificities within plant members of GH27 and GH36.…”

Section: Enzymes: Structure–function Relationshipsmentioning

confidence: 99%

Multifunctional fructans and raffinose family oligosaccharides

Ende¹

2013

Front. Plant Sci.

182

116

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Fructans and raffinose family oligosaccharides (RFOs) are the two most important classes of water-soluble carbohydrates in plants. Recent progress is summarized on their metabolism (and regulation) and on their functions in plants and in food (prebiotics, antioxidants). Interest has shifted from the classic inulin-type fructans to more complex fructans. Similarly, alternative RFOs were discovered next to the classic RFOs. Considerable progress has been made in the understanding of structure–function relationships among different kinds of plant fructan metabolizing enzymes. This helps to understand their evolution from (invertase) ancestors, and the evolution and role of so-called “defective invertases.” Both fructans and RFOs can act as reserve carbohydrates, membrane stabilizers and stress tolerance mediators. Fructan metabolism can also play a role in osmoregulation (e.g., flower opening) and source–sink relationships. Here, two novel emerging roles are highlighted. First, fructans and RFOs may contribute to overall cellular reactive oxygen species (ROS) homeostasis by specific ROS scavenging processes in the vicinity of organellar membranes (e.g., vacuole, chloroplasts). Second, it is hypothesized that small fructans and RFOs act as phloem-mobile signaling compounds under stress. It is speculated that such underlying antioxidant and oligosaccharide signaling mechanisms contribute to disease prevention in plants as well as in animals and in humans.

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“…suggested that PhGal31A and PsGal31A were the first GH31 enzymes with activity for α-galactosides, and have different substrate specificity than that of the characterized α-galactosidases, which hydrolyze α(1-3)-, α(1-4)-, and α(1-6)-galactoside linkages [52][53][54][55].…”

Section: General Properties and Substrate Specificities Of Phgal31a Amentioning

confidence: 99%

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31

Miyazaki

Ishizaki

Ichikawa

et al. 2015

Biochemical Journal

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Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family.

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The Molecular Mechanism of Thermostable α-Galactosidases AgaA and AgaB Explained by X-ray Crystallography and Mutational Studies

Cited by 40 publications

References 43 publications

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Multifunctional fructans and raffinose family oligosaccharides

Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31

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