Glycoside Hydrolase Family 88

This page is currently under construction. This means that the Responsible Curator has deemed that the page's content is not quite up to CAZypedia's standards for full public consumption. All information should be considered to be under revision and may be subject to major changes.

• Author: ^^^Seino Jongkees^^^
• Responsible Curator: ^^^Steve Withers^^^

---

Glycoside Hydrolase Family GH88
Clan	none
Mechanism	N/A
Active site residues	not known
CAZy DB link
https://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 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

Error fetching PMID 16893885:
Error fetching PMID 22047074:
Error fetching PMID 10441389:
Error fetching PMID 12044176:
Error fetching PMID 12729728:
Error fetching PMID 19416976:
Error fetching PMID 21147778:
Error fetching PMID 22311922:
Error fetching PMID 24403065:
Error fetching PMID 24227702:
Error fetching PMID 16781735:
Error fetching PMID 16870154:
Error fetching PMID 16332065:
Error fetching PMID 3096349:
Error fetching PMID 9195886:
Error fetching PMID 10978344:
Error fetching PMID 8952475:
Error fetching PMID 3278958:
Error fetching PMID 1731996:
Error fetching PMID 16917793:
Error fetching PMID 24573682:
Error fetching PMID 15148314:
Error fetching PMID 16630576:

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 |

22. Koshland DE Jr: Stereochemistry and the mechanism of enzyme reactions. Biol Rev 1953, 28:416-436.