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Glycoside Hydrolase Family 105

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Glycoside Hydrolase Family GH105
Clan none
Mechanism N/A
Active site residues known
CAZy DB link

Substrate specificities

GH105 enzymes are a class of unsaturated glucuronyl/galacturonyl hydrolases found mainly in bacteria, but a few fungal and a handful of archaeal enzymes have also been annotated [1]. Much like the Glycoside Hydrolase Family 88, enzymes from GH105 perform hydrolysis via a hydration of the double bond between the C-4 and C-5 carbons of the terminal monosaccharide of their substrates [2, 3]. Enzymes from GH105 have been organized into three subgroups: unsaturated rhamnogalacturonidases, d-4,5-unsaturated β-glucuronyl hydrolases, and d-4,5-unsaturated α-galacturonidases. The unifying feature shared between these substrates is the presence of the non-reducing monosaccharide 4-deoxy-L-threo-hex-4-enopyranuronosyl that binds at the -1 active site of the enzymes and is linked to the +1 sugar via its anomeric C-1 carbon. The 4-deoxy-L-threo-hex-4-enopyranuronosyl saccharide is defined as ΔGal or ΔGlc depending on whether it assumes an α- or β- configuration, respectively. In degradable substrates, the sugar present at the +1 position can be linked via its C-2, C-4, or C-6 carbon, given the substrate preference of individual enzymes [2, 4]. Some of the various carbohydrate sources targeted by GH105 enzymes include: rhamnogalacturonan-I, ulvan, and the arabinogalactan decoration on certain cell wall proteins [2, 5, 6, 7].

Kinetics and Mechanism

GH105 enzymes do not act via a typical Koshland retaining or inverting mechanism [8], rather the current proposed mechanism of action for these enzymes is hydrolysis through syn-hydration of the double bond between the C-4 and C-5 carbons of the enopyranuronosyl residue of their substrate [5]. This hydration reaction forms a hemiketal that undergoes spontaneous rearrangement to form an intermediate hemiacetyl, which undergoes further rearrangement resulting in the breakage of the bond to the neighbouring saccharide (at the +1 subsite of the enzyme) of the polymer. This mechanism was initially theorized based on the oligosaccharide and amino acid arrangement in a substrate-bound crystal structure [6], but has been confirmed through kinetic isotope effects and NMR analysis in the highly related unsaturated glucuronyl hydrolase GH88 family [3, 9].

The kinetics for three enzymes from the GH105 family have been determined, two from Bacillus subtilis and one from Bacteriodes thetaiotaomicron. YteR from B. subtilis was found to have a kcat and KM of 0.2±0.011s-1 and 100±14μM, respectively, against the substrate ΔGal-Rha; in contrast, YesR was found to have much higher values for both these kinetic parameters, 13.9±0.7s-1 and 719±75μM for kcat and KM, respectively, with the same substrate [6]. BT3687 from B. thetaiotaomicron was determined to have a kcat of 0.59±0.057s-1 and a KM of 71.87±12.51μM against the substrate ΔGlc-GlcNAc [2].

Although it is atypical for a glycoside hydrolase family to contain enzymes capable of degrading both α- or β-linked substrates, this has also been observed in other families that deviate significantly from typical acid-base mechanisms (eg. GH3, GH4) [10].

Catalytic Residues

A single aspartate residue has been proposed to be responsible for the hydration reaction based on substrate-complexed X-ray crystal structures, sequence conservation, and site-directed mutagenesis [5, 6]. The first enzyme classified into the GH105 family (PDB ID 1NC5 from B. subtilis) was originally predicted to be a lyase based on 65% amino acid sequence similarity and over 60% matching secondary-structure characteristics with an N-acyl-D-glucosamine 2-epimerase [4]. Following sequence comparison to a GH88 hydrolase (a B. subtilis UGL enzyme), and additional functional characterization, 1NC5 was determined to possess unsaturated galacturonyl hydrolase activity [6]. A conserved aspartate residue (D143), was found to be the most likely candidate for initiating the hydration reaction, while a second conserved residue, histidine (H189), serves to correctly position a water molecule for deprotonation and addition to the C-5 carbon of the monosaccharide in the enzyme's -1 subsite [6]. Based on sequence alignment and structural analysis, an arginine residue may take the place of this histidine residue in some GH105 enzymes (e.g. PDB ID 4CE7 and PDB ID 5NOA) [2, 7, 11].

Three-dimensional structures

A number of crystal structures of GH105 unsaturated glucuronyl hydrolases expressed in bacteria have been solved, including several structures from B. subtilis [4, 5, 6], and B. thetaiotaomicron [2, 7, 12], as well as one each from Bacteriodes vulgatus (PDB ID 4Q88), Clostridium acetobutylicum [13], Klebsiella pneumoniae (PDB ID 3PMM), and Salmonella enterica (PDB ID 3QWT). A single enzyme from the fungus Thielavia terrestris has also been solved (PDB ID 4XUV). All of these enzymes share an (α/α)6-barrel structure (also similar to that of the related GH88 enzymes), with the main differences being seen in the structure of the loop region that determines the architecture of the binding site. At the bottom of the active site pocket is a conserved WxRxxGW motif, with the tryptophan and arginine residues forming a pocket that engages the carboxyl group on the ΔGal/Glc monosaccharide of the -1 subsite [5]. While several residues may be conserved in sequence and position at the -1 subsite, the +1 subsite is much more variable, which likely accounts for the ability of this enzyme family to catalyze the hydrolysis of polysaccharides containing α- or β-bonds linked to the C-2, -4, or -6 carbon of the +1 saccharide [7].

Family Firsts

First stereochemistry determination
Crystal structure of substrate-bound B. subtilis YteR unsaturated rhamnogalacturonan hydrolase in 2006. Functional analysis of this enzyme detected loss of a C=C bond compared to a detectable increase in α-keto acid following enzyme-substrate incubation [6].
First catalytic residue identification
Crystal structure analysis of the B. subtilis YteR enzyme complexed with a ΔGlc-GalNac substrate analog suggested Asp143 is responsible for initiating the hydration reaction and kinetic assessment of a D143N mutant of YteR showed complete loss of catalytic activity [6].
First evidence of hydration-based mechanism
While the mechanism of GH105 enzymes has not been fully described, the mechanism of the unsaturated glucuronyl hydrolase (UGL) from Clostridium perfringens (a closely-related GH88 protein) was determined via NMR using a methyl ketal intermediate analogue and monitoring of the reaction product during enzyme-substrate incubation in D2O [3].
First 3-D structure
The 1.6Å crystal structure of the B. subtilis protein YteR, initially predicted to be a lyase-type enzyme, was reported in 2005 [4].


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  2. Munoz-Munoz J, Cartmell A, Terrapon N, Baslé A, Henrissat B, and Gilbert HJ. (2017) An evolutionarily distinct family of polysaccharide lyases removes rhamnose capping of complex arabinogalactan proteins. J Biol Chem. 292, 13271-13283. DOI:10.1074/jbc.M117.794578 | PubMed ID:28637865 | HubMed [Munoz-Munoz2017]
  3. Jongkees SA and Withers SG. (2011) Glycoside cleavage by a new mechanism in unsaturated glucuronyl hydrolases. J Am Chem Soc. 133, 19334-7. DOI:10.1021/ja209067v | PubMed ID:22047074 | HubMed [Jongkees2011]
  4. Zhang R, Minh T, Lezondra L, Korolev S, Moy SF, Collart F, and Joachimiak A. (2005) 1.6 A crystal structure of YteR protein from Bacillus subtilis, a predicted lyase. Proteins. 60, 561-5. DOI:10.1002/prot.20410 | PubMed ID:15906318 | HubMed [Zhang2009]
  5. Itoh T, Ochiai A, Mikami B, Hashimoto W, and Murata K. (2006) Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate. Biochem Biophys Res Commun. 347, 1021-9. DOI:10.1016/j.bbrc.2006.07.034 | PubMed ID:16870154 | HubMed [Itoh2006]
  6. Itoh T, Ochiai A, Mikami B, Hashimoto W, and Murata K. (2006) 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. J Mol Biol. 360, 573-85. DOI:10.1016/j.jmb.2006.04.047 | PubMed ID:16781735 | HubMed [Itoh2006-1]
  7. Collén PN, Jeudy A, Sassi JF, Groisillier A, Czjzek M, Coutinho PM, and Helbert W. (2014) A novel unsaturated β-glucuronyl hydrolase involved in ulvan degradation unveils the versatility of stereochemistry requirements in family GH105. J Biol Chem. 289, 6199-211. DOI:10.1074/jbc.M113.537480 | PubMed ID:24407291 | HubMed [Collen2014]
  8. Koshland, D.E. (1953) Stereochemistry and the Mechanism of Enzymatic Reactions. Biological Reviews, vol. 28, no. 4., pp. 416-436. DOI:10.1111/j.1469-185X.1953.tb01386.x.
  9. Jongkees SA, Yoo H, and Withers SG. (2014) Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens. J Biol Chem. 289, 11385-95. DOI:10.1074/jbc.M113.545293 | PubMed ID:24573682 | HubMed [Jongkees2014]
  10. Rye CS and Withers SG. (2000) Glycosidase mechanisms. Curr Opin Chem Biol. 4, 573-80. DOI:10.1016/s1367-5931(00)00135-6 | PubMed ID:11006547 | HubMed [Rye2000]
  11. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, and Ferrin TE. (2004) UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem. 25, 1605-12. DOI:10.1002/jcc.20084 | PubMed ID:15264254 | HubMed [Pettersen2004]
  12. Joint Center for Structural Genomics(JCSG) (2009) Crystal structure of Putative glycosyl hydrolase (NP_813087.1) from BACTEROIDES THETAIOTAOMICRON VPI-5482 at 1.80 A resolution. RCSB Protein Data Bank. [1].
  13. Germane KL, Servinsky MD, Gerlach ES, Sund CJ, and Hurley MM. (2015) Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105. Acta Crystallogr F Struct Biol Commun. 71, 1100-8. DOI:10.1107/S2053230X15012121 | PubMed ID:26249707 | HubMed [Germane2015]
All Medline abstracts: PubMed | HubMed