Glycoside Hydrolase Family 88

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• Author: Seino Jongkees
• Responsible Curator: Steve Withers

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Glycoside Hydrolase Family GH88
Clan	none
Mechanism	N/A
Active site residues	not known
CAZy DB link
http://www.cazy.org/GH88.html

Substrate specificities

GH88 enzymes are unsaturated glucuronyl hydrolases, and are predominantly found expressed in bacteria, although a small number have been annotated in archaea and fungi [1]. These enzymes are atypical in that they hydrolyse their substrates through hydration of a double bond between carbons 4 and 5 of the non-reducing terminal sugar of their substrates [2, 3]. Substrates for GH88 are derived for the most part from the activity of Polysaccharide Lyases on glycosaminoglycans, and so are β-1,3- or β-1,4-linked (before elimination; lyase cleavage changes the reference stereocenter, leading to the products being α linked, but the anomeric bond does not change). The preferred pattern of sulphation in substrates varies with the source organism [4, 5, 6, 7, 8, 9, 10, 11], and a flexible loop adjacent to the +2 subsite has been identified as an important determinant of this preference [8]. The related family GH105 contains enzymes that cleave alpha linked substrates, typically working on substrates derived from rhamnogalacturonans [2, 12]. Aside from glycosaminoglycans, unsaturated uronic acid-containing oligosaccharides from gellan and xanthan have also been identified as substrates [4, 6].

GH88 from Clostridium perfringens has also been shown to be capable of acting on several unusual substrates [3]. It is able to hydrate a C-glycosidic substrate analogue, and hydrolyses both the thioglycoside analogue and the alternate anomer of phenyl unsaturated glucuronic acid. Previously, only GH84 [13], GH1 [14, 15, 16, 17, 18, 19], and GH4 [20] had been shown to hydrolyse thioglycosides. UGL is able to hydrolyse a range of synthetic substrates with aromatic leaving groups, as well as unsaturated glucuronyl fluoride with both anomeric stereochemistries [11], with k_cat decreasing as electron withdrawing ability increases. Finally, substrates with the hydroxyl group on carbon 2 replaced appear to be turned over very poorly [5, 11, 21], although in some cases sulfation appears to at least be partially tolerated in this position [4, 6, 7, 10].

Kinetics and Mechanism

The GH88 family enzymes do not follow a classical Koshland inverting mechanism or retaining mechanism [22]. Enzymes in this family instead are believed to trigger hydrolysis by hydration of the double bond between carbons 4 and 5 in their substrates. This hydration product, a hemiketal, then undergoes a series of rearrangements — forming first an intermediate hemiacetal, then loss of the anomeric substituent to give an open chain product, which can then be hydrated in water. This mechanism was initially proposed based on catalytic residue placement in a substrate-bound crystal structure [2], and subsequently confirmed by kinetic isotope effect and NMR data [3]. The initial hydration is a syn addition of water to the double bond, while carbon 1 cannot be said to have a stereochemical outcome, as it is an aldehyde in the first-formed product and immediately forms a mixture of anomers [3]. The rearrangements from hemiketal to open chain product have been suggested to be catalysed by the enzyme on the basis of NMR studies looking for the first detectable products under high enzyme concentration conditions, wherein no intermediates were seen to accumulate in solution [21].

Based on kinetic data, the hydration step of the mechanism appears to proceed through a short-lived intermediate. This intermediate has been suggested to be a pyranose-ring-opened structure with a C4 ketone and a C1-C2 epoxide, on the basis of kinetic isotope effects, C2 hydroxyl group substitutions, and catalytic residue placement [21]. However, there is not yet any direct evidence for this. The overall rate-limiting step of this mechanism is believed to be the breakdown of this intermediate of the hydration process, on the basis of kinetic isotope effects [21] and consistent with LFER data [11].

Catalytic Residues

Catalytic residues have been proposed based on sequence conservation, X-ray crystallography, and site-directed mutagenesis studies [2, 23]. These amino acids, a pair of aspartate or glutamate residues, were initially thought to be involved in an inverting type mechanism, on the basis of their separation distance [23]. Subsequent solving of x-ray crystal structures of a catalytically-inactive mutant with substrates bound led to the proposal of the hydration-initiated mechanism outlined above [2]. However, while two carboxylate-containing residues are present in the active site, and mutation of either to the corresponding amide side-chain gives a mutant with near-negligible activity, only one of these has a clear role. Aspartate number 149 in Bacillus sp. GL1 UGL is situated 2.9 Å from the substrate carbon 4, and plays the role of a catalytic acid to protonate the C4-C5 double bond in the substrate, thereby initiating hydration. Aspartate 88 in the same enzyme, as the asparagine mutant, is situated adjacent to the hydroxyl groups on carbons 2 and 3, at around 2.4 Å from each. This residue has no proposed role in a direct hydration mechanism that could account for its apparent importance in catalysis. However, it has been proposed that this residue may be important for the formation of a transient intermediate in the hydration step [21].

Three-dimensional structures

Several x-ray crystal structures of unsaturated glucuronyl hydrolases have been solved, from Bacillus sp. GL1[2, 12, 23], several Streptococcus species [7, 8], and Pedobacter heparinus [10]. The substrate-bound crystal structure from Bacillus sp GL1 was responsible for the suggestion of a hydration-initiated hydrolysis mechanism [2]. Much structural work has focussed on the determinants of substrate specificity, particularly the discrimination of unsaturated glucuronides from different glycosaminoglycan sources. Comparison of the Streptococcal and Bacillus structures identified a flexible loop involved in recognition of sulfation patterns in the +1 subsite [8].

Family Firsts

First stereochemistry determination

Bacillus sp. GL1 UGL (unsaturated glucuronyl hydrolase), enzyme alone [23]

Bacillus sp. GL1 UGL (unsaturated glucuronyl hydrolase), substrate bound [2]

First catalytic residue determination

Bacillus sp. GL1 UGL (unsaturated glucuronyl hydrolase), via x-ray crystal structure [23]

First stereochemistry determination (hydration)

Clostridium perfringens UGL (unsaturated glucuronyl hydrolase), via NMR of methyl ketal intermediate analogue and product of reaction in deuterated water [3]

First evidence for intermediate in hydration

Clostridium perfringens UGL (unsaturated glucuronyl hydrolase), from kinetic isotope effects and substrate analogues [21]

References

1. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, and Henrissat B. (2009). The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. Nucleic Acids Res. 2009;37(Database issue):D233-8. DOI:10.1093/nar/gkn663 |
2. 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. 2006;360(3):573-85. DOI:10.1016/j.jmb.2006.04.047 |
2. Itoh T, Hashimoto W, Mikami B, and Murata K. (2006). Crystal structure of unsaturated glucuronyl hydrolase complexed with substrate: molecular insights into its catalytic reaction mechanism. J Biol Chem. 2006;281(40):29807-16. DOI:10.1074/jbc.M604975200 |
3. Jongkees SA and Withers SG. (2011). Glycoside cleavage by a new mechanism in unsaturated glucuronyl hydrolases. J Am Chem Soc. 2011;133(48):19334-7. DOI:10.1021/ja209067v |
4. Hashimoto W, Kobayashi E, Nankai H, Sato N, Miya T, Kawai S, and Murata K. (1999). Unsaturated glucuronyl hydrolase of Bacillus sp. GL1: novel enzyme prerequisite for metabolism of unsaturated oligosaccharides produced by polysaccharide lyases. Arch Biochem Biophys. 1999;368(2):367-74. DOI:10.1006/abbi.1999.1305 |
5. Myette JR, Shriver Z, Kiziltepe T, McLean MW, Venkataraman G, and Sasisekharan R. (2002). Molecular cloning of the heparin/heparan sulfate delta 4,5 unsaturated glycuronidase from Flavobacterium heparinum, its recombinant expression in Escherichia coli, and biochemical determination of its unique substrate specificity. Biochemistry. 2002;41(23):7424-34. DOI:10.1021/bi012147o |
6. Mori S, Akao S, Nankai H, Hashimoto W, Mikami B, and Murata K. (2003). A novel member of glycoside hydrolase family 88: overexpression, purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1. Protein Expr Purif. 2003;29(1):77-84. DOI:10.1016/s1046-5928(03)00019-6 |
7. Maruyama Y, Nakamichi Y, Itoh T, Mikami B, Hashimoto W, and Murata K. (2009). Substrate specificity of streptococcal unsaturated glucuronyl hydrolases for sulfated glycosaminoglycan. J Biol Chem. 2009;284(27):18059-69. DOI:10.1074/jbc.M109.005660 |
8. Nakamichi Y, Maruyama Y, Mikami B, Hashimoto W, and Murata K. (2011). Structural determinants in streptococcal unsaturated glucuronyl hydrolase for recognition of glycosaminoglycan sulfate groups. J Biol Chem. 2011;286(8):6262-71. DOI:10.1074/jbc.M110.182618 |
9. Marion C, Stewart JM, Tazi MF, Burnaugh AM, Linke CM, Woodiga SA, and King SJ. (2012). Streptococcus pneumoniae can utilize multiple sources of hyaluronic acid for growth. Infect Immun. 2012;80(4):1390-8. DOI:10.1128/IAI.05756-11 |
10. Nakamichi Y, Mikami B, Murata K, and Hashimoto W. (2014). Crystal structure of a bacterial unsaturated glucuronyl hydrolase with specificity for heparin. J Biol Chem. 2014;289(8):4787-97. DOI:10.1074/jbc.M113.522573 |
11. Jongkees SA, Yoo H, and Withers SG. (2014). Mechanistic insights from substrate preference in unsaturated glucuronyl hydrolase. Chembiochem. 2014;15(1):124-34. DOI:10.1002/cbic.201300547 |
12. Itoh T, Hashimoto W, Mikami B, and Murata K. (2006). Substrate recognition by unsaturated glucuronyl hydrolase from Bacillus sp. GL1. Biochem Biophys Res Commun. 2006;344(1):253-62. DOI:10.1016/j.bbrc.2006.03.141 |
12. Itoh T, Ochiai A, Mikami B, Hashimoto W, and Murata K. (2006). Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate. Biochem Biophys Res Commun. 2006;347(4):1021-9. DOI:10.1016/j.bbrc.2006.07.034 |
13. Macauley MS, Stubbs KA, and Vocadlo DJ. (2005). O-GlcNAcase catalyzes cleavage of thioglycosides without general acid catalysis. J Am Chem Soc. 2005;127(49):17202-3. DOI:10.1021/ja0567687 |
14. Day AG and Withers SG. (1986). The purification and characterization of a beta-glucosidase from Alcaligenes faecalis. Biochem Cell Biol. 1986;64(9):914-22. DOI:10.1139/o86-122 |
15. Burmeister WP, Cottaz S, Driguez H, Iori R, Palmieri S, and Henrissat B. (1997). The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase. Structure. 1997;5(5):663-75. DOI:10.1016/s0969-2126(97)00221-9 |
16. Burmeister WP, Cottaz S, Rollin P, Vasella A, and Henrissat B. (2000). High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. J Biol Chem. 2000;275(50):39385-93. DOI:10.1074/jbc.M006796200 |
17. Cottaz S, Henrissat B, and Driguez H. (1996). Mechanism-based inhibition and stereochemistry of glucosinolate hydrolysis by myrosinase. Biochemistry. 1996;35(48):15256-9. DOI:10.1021/bi9622480 |
18. McDanell R, McLean AE, Hanley AB, Heaney RK, and Fenwick GR. (1988). Chemical and biological properties of indole glucosinolates (glucobrassicins): a review. Food Chem Toxicol. 1988;26(1):59-70. DOI:10.1016/0278-6915(88)90042-7 |
19. Xue JP, Lenman M, Falk A, and Rask L. (1992). The glucosinolate-degrading enzyme myrosinase in Brassicaceae is encoded by a gene family. Plant Mol Biol. 1992;18(2):387-98. DOI:10.1007/BF00034965 |
20. Yip VL and Withers SG. (2006). Family 4 glycosidases carry out efficient hydrolysis of thioglycosides by an alpha,beta-elimination mechanism. Angew Chem Int Ed Engl. 2006;45(37):6179-82. DOI:10.1002/anie.200601421 |
21. Jongkees SAK, Yoo H, and Withers SG. (2014). Mechanistic investigations of unsaturated glucuronyl hydrolase from Clostridium perfringens. J Biol Chem. 2014;289(16):11385-11395. DOI:10.1074/jbc.M113.545293 |

22. Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436. DOI:10.1111/j.1469-185X.1953.tb01386.x

23. Itoh T, Akao S, Hashimoto W, Mikami B, and Murata K. (2004). Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp. GL1 at 1.8 A resolution. J Biol Chem. 2004;279(30):31804-12. DOI:10.1074/jbc.M403288200 |