Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases

Pitson, Stuart M.; Voragen, A.G.J.; Beldman, G.

doi:10.1016/s0014-5793(96)01153-2

Cited by 63 publications

(47 citation statements)

References 48 publications

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“…To confirm this assignment, the new reaction product was purified and characterized by 1 H NMR. The observed multiplicity of the anomeric proton (doublet at 4.74 ppm), and its coupling constant (J 1,2 ϭ 1.6 Hz), is consistent with the product being methyl ␣-L-arabinofuranoside (15).…”

Section: Resultssupporting

confidence: 59%

“…Interestingly, further incubation of the reaction resulted in the hydrolysis of the new product by the enzyme, yielding arabinofuranoside and methanol. The hydrolysis of the methyl ␣-L-arabinofuranoside product was also observed in the case of arabinofuranosidase A from Aspergillus niger (15). This simple TLC-based approach to determine the stereochemical course of hydrolysis can be easily applied to other glycoside hydrolases with unknown stereochemistry.…”

Section: Resultsmentioning

confidence: 80%

“…In the case of arabinofuranosidases, the arabinofuranoside undergoes fast mutarotation forming the four arabinose tautomers: ␣-and ␤-L-arabinofuranosides and ␣-and ␤-L-arabinopyranosides. To overcome this kind of experimental limitation, the stereochemical courses of the ␣-L-arabinofuranosidases from GH51 and GH54 were determined by monitoring the hydrolysis of 4-NPAF in the presence of 2.5 M methanol with 1 H NMR (15). For many retaining glycoside hydrolases, methanol can act as an external nucleophile, replacing the water molecule, and the resulting methyl ␣-L-arabinofuranoside is stable enough to be characterized by 1 H NMR analysis.…”

Section: Resultsmentioning

confidence: 99%

“…In the second step (deglycosylation), the acid-base residue, acting this time as a general base, activates a water molecule that attacks the anomeric center of the glycosyl-enzyme intermediate from the same direction of the original bond, liberating the free sugar with an overall retention of the anomeric configuration (13,14). Both catalytic mechanisms are found in the different ␣-L-arabinofuranosidases families: GH51 and GH54 were shown to cleave the glycosidic bond with a retention of the anomeric configuration (15); GH43, which includes ␣-L-arabinofuranosidases as well as ␤-D-xylosidases, was shown to work via the inverting mechanism (15), whereas the stereochemistry of GH62 is not yet characterized. Currently there are 32 sequences of ␣-L-arabinofuranosidases classified as GH51 glycoside hydrolases, 2 and, although much work was done on the substrate specificities of these enzymes, there are only a few studies concerning the biochemical mechanism and catalytic properties of this family (17)(18)(19).…”

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confidence: 99%

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Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

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Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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“…Enzymes in CAZy that hydrolyze polysaccharides are named after their substrate, GH family, the initials of the organism of origin and given a letter to indicate the order in which they were first reported (Henrissat et al, 1998 1,5--L-arabinanase (AnArb43A) were shown by NMR to use a single displacement mechanism resulting in products with inverted anomeric configuration (Pitson et al, 1996;Wilkens et al, 2016).…”

Section: Classification Of -L-arabinofuranosidasesmentioning

confidence: 99%

GH62 arabinofuranosidases: Structure, function and applications

Wilkens

Andersen

Dumon

et al. 2017

Biotechnology Advances

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Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. --1,3 arabinofuranosyl specific -L-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release -L-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies -L-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively -L-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 -L-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.3

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Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity

et al. 2020

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Biosynthesis of the peptidyl nucleoside antifungal agent blasticidin S in Streptomyces griseochromogenes requires the hydrolytic function of a nucleotide hydrolase, BlsM, to excise the free cytosine from the 5′‐monophosphate cytosine nucleotide. In addition to its hydrolytic activity, interestingly, BlsM has also been shown to possess a novel cytidine deaminase activity, converting cytidine, and deoxycytidine to uridine and deoxyuridine. To gain insight into the substrate specificity of BlsM and the mechanism by which it performs these dual function, the solution structure of BlsM was determined by multi‐dimensional nuclear magnetic resonance approaches. BlsM displays a nucleoside deoxyribosyltransferase‐like dimeric topology, with each monomer consisting of a five‐stranded β‐sheet that is sandwiched by five α‐helixes. Compared with the purine nucleotide hydrolase RCL, each monomer of BlsM has a smaller active site pocket, enclosed by a group of conserved hydrophobic residues from both monomers. The smaller size of active site is consistent with its substrate specificity for a pyrimidine, whereas a much more open active site, as in RCL might be required to accommodate a larger purine ring. In addition, BlsM confers its substrate specificity for a ribosyl‐nucleotide through a key residue, Phe19. When mutated to a tyrosine, F19Y reverses its substrate preference. While significantly impaired in its hydrolytic capability, F19Y exhibited a pronounced deaminase activity on CMP, presumably due to an altered substrate orientation as a result of a steric clash between the 2′‐hydroxyl of CMP and the ζ‐OH group of F19Y. Finally, Glu105 appears to be critical for the dual function of BlsM.

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Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases

Cited by 63 publications

References 48 publications

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

GH62 arabinofuranosidases: Structure, function and applications

Solution structure of the nucleotide hydrolase BlsM: Implication of its substrate specificity

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