RGI, degrading enzymes are active on the RGI backbone of pectin and are thus strictly specific for cleaving bonds in the repetitive [(1->2)-alpha-L-Rhap-(1->4)-alpha-D-GalpA-(1->2)] structure
postulation of the enzyme reaction mechanism: YteR triggers the hydration of vinyl ether group in 4-deoxy-beta-L-threo-hex-4-enopyranuronose (DELTAGalA), but not of glycoside bond, by using Asp143 as a general acid and base catalyst. Asp143 donates proton to the double bond of 4-deoxy-beta-L-threo-hex-4-enopyranuronose as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of 4-deoxy-beta-L-threo-hex-4-enopyranuronose
postulation of the enzyme reaction mechanism: YteR triggers the hydration of vinyl ether group in 4-deoxy-beta-L-threo-hex-4-enopyranuronose (DELTAGalA), but not of glycoside bond, by using Asp143 as a general acid and base catalyst. Asp143 donates proton to the double bond of 4-deoxy-beta-L-threo-hex-4-enopyranuronose as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of 4-deoxy-beta-L-threo-hex-4-enopyranuronose
URGH is active only on RGI oligomers with alpha-DELTA4,5-unsaturated-GalpA (i.e. 2-O-(4-deoxy-beta-L-threo-hex-4-enopyra-nuronosyl)) at the non-reducing end. The URGH activity catalyses the cleavage of the alpha-(1->2) glycosidic bond between the DELTA4,5-unsaturated-GalpA and L-Rhap releasing single unsaturated DELTAGalpA (5-dehydro-4-deoxy-D-galacturonate)
URGH is active only on RGI oligomers with alpha-DELTA4,5-unsaturated-GalpA (i.e. 2-O-(4-deoxy-beta-L-threo-hex-4-enopyra-nuronosyl)) at the non-reducing end. The URGH activity catalyses the cleavage of the alpha-(1->2) glycosidic bond between the DELTA4,5-unsaturated-GalpA and L-Rhap releasing single unsaturated DELTAGalpA (5-dehydro-4-deoxy-D-galacturonate)
the degradation of the polygalacturonan (PGA) and rhamnogalacturonan-I (RG-I) backbones of pectin. In both instances the unsaturated GalA may spontaneously open to 4-deoxy-L-threo-5-hexosulose-uronate, reaction pathways, overview. The suggested mechanism for both families, the unsaturated rhamnogalacturonyl hydrolase (EC 3.2.1.172) and the unsaturated glucuronyl hydrolase (EC 3.2.1.179), requires hydration at the C=C bond of the unsaturated sugar, resulting in glycosidic cleavage. Substrate specificity in the GH105 family correlates with the loops surrounding the active site
RGI, degrading enzymes are active on the RGI backbone of pectin and are thus strictly specific for cleaving bonds in the repetitive [(1->2)-alpha-L-Rhap-(1->4)-alpha-D-GalpA-(1->2)] structure
the enzyme expression is induced threefold and sixfold during growth on pectin and polygalacturonic acid, respectively, when compared with growth on glucose
Clostridium acetobutylicum ATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase (EC 3.2.1.172) and the unsaturated glucuronyl hydrolase (EC 3.2.1.179) from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggest that the enzyme may have a slightly different substrate specificity from that of YteR
phylogenetic and structural traits of RGI hydrolases, overview. The enzyme belongs to the glycosyl hydrolase family 105, GH105. Enzyme URGH from GH105 has a alpha/alpha double toroid structure with six-a-hairpins arranged in a double helical barrel
phylogenetic and structural traits of RGI hydrolases, overview. The enzyme belongs to the glycosyl hydrolase family 105, GH105. The URGH from GH105 has a alpha/alpha double toroid structure with six-a-hairpins arranged in a double helical barrel
in the active site of YteR Asp143 is most likely functioning as proton donor. Residues Asp88 and Tyr41 may modulate the pKa of Asp143 in YteR thereby inducing the lower pH optimum of this enzyme
in the active site of YteR Asp143 is most likely functioning as proton donor. Residues Asp88 and Tyr41 may modulate the pKa of Asp143 in YteR thereby inducing the lower pH optimum of this enzyme
the protein adopts a six-(alpha/alpha)-hairpin barrel fold with a small two-stranded beta-sheet and helix overlaying the end of the barrel near the active pocket. The CA_C0359 structure closely adopts the fold of family GH105 proteins
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, crystal structure of YteR complexed with unsaturated chondroitin disaccharide (substrate of bacterial unsaturated glucuronyl hydrolase). The substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively
hanging drop vapor diffusion method, structure of D143N in complex with unsaturated rhamnogalacturonan disaccharide substrate 2-O-(4-deoxy-beta-L-threo-hex-4-enopyranuronosyl)-alpha-L-rhamnopyranose is determined at 1.9 A resolution by X-ray crystallography
partially purified recombinnat enzyme, hanging-drop vapor-diffusion method, mixing 0.002 ml 7.5 mg/ml protein solution with 0.002 ml reservoir solution consisting of 0.1 M Tris, pH 7.75, 16% w/v PEG 4000, 21°C, X-ray diffraction structure determination and analysis at 1.6 A resolution, modelling
gene CA_C0359, recombinant expression of the intein-fused enzyme in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli TOP10, transcriptional analysis using quantitative PCR
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme expression is induced threefold and sixfold during growth on pectin and polygalacturonic acid, respectively, when compared with growth on glucose
Itoh, T.; Ochiai, A.; Mikami, B.; Hashimoto, W.; Murata, K.
A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide