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Glycoside Hydrolase Family 84

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Glycoside Hydrolase Family GH84
Clan none
Mechanism retaining
Active site residues known
CAZy DB link
http://www.cazy.org/GH84.html


Substrate specificities

GH84 contains β-N-acetylglucosaminidases and β-N-acetylhyaluronidase activities. Human O-GlcNAcase is a nucleocytoplasmic enzyme whose in vivo targets are glycoprotein serine and threonine residues modified by a single β-linked GlcNAc residue. In contrast to the β-hexosaminidases of GH20 a relaxed specificity for substitutions of the N-acyl group is observed with residues significantly more bulky than the N-acyl group being tolerated.[1]


Kinetics and Mechanism

Members of GH84 utilize a mechanism of neighbouring group participation, this originally being established through the use of free-energy relationship-based studies of human O-GlNAcase.[1] More recent studies of this enzyme have investigated variations in rates of reaction (V/K) with both nucleophile and leaving group structures.[2] For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on V/K for leaving groups with a pKa below 11 (with the chemical step rate-determining for substrates with higher pKa values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (DN*AN) mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a pKa greater than approximately 7; a concerted (ANDN) mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower pKas (consistent with prior studies of S-glucosaminide hydrolysis[3]).

Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yield potent inhibitors of GH84 enzymes. These include "NAG-thiazolines",REF, PUGNAc (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate),[4] GlcNAcstatin, [5, 6, 6, 7, 8, 9, 10, 10, 10, 10, 11, 11, 12, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 32, 33, 34, 35, 36, 37, 38, 39, 40, 40, 41, 42, 43, 44, 45, 46, 47, 48] Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human O-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their N-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate.[49] As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based)[50] transition state analogues. Substrate distortion.[2, 51]

A truncated, nuclear-localized isoform of human O-GlcNAcase lacking the putative C-terminal histone acetyl transferase domain retains similar kinetic properties and inhibitory patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the N-terminal domain.[52]


Catalytic Residues

Studies of two mutants of human O-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme.[53]

The mutant Asp175Ala displayed marked reductions in activity (V and (V/K)) towards aryl N-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both O-aryl and S-aryl N-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human O-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results identify Asp175 as the general acid catalyst.

The mutant Asp174Ala showed decreased activity towards O-aryl N-acetylglucosaminides possessing good leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the N-acyl nucleophile.


Three-dimensional structures

The reported crystallization of Clostridium perfringens NagJ[54] was followed by solved structures for that enzyme[55] and Bacteroides thetaiotaomicron β-hexosaminidase[55]. In common with the chitinases of family GH18 and the exo-acting β-hexosaminidases of GH20 the catalytic domain is a (βα)8-barrel structure. The structure of human O-GlcNAcase has not been solved but Bacteroides thetaiotaomicron β-hexosaminidase was originally reported as a good structural mimic of both the active site and catalytic domain of human O-GlcNAcase. More recently the structure of the GH84 β-hexosaminidase from Oceanicola granulosus has been solved and shown to possess an improved sequence identity with the catalytic domain of the human enzyme.[56] Furthermore, the C-terminal domain of this protein also displays notable sequence identity with the spacer domain (separating the domains possessing O-GlcNAcase and histone acetyltransferase activities) of the human enzyme.

Defining the conformational itinerary of a GH84 enzyme. Azepane (i) binds with a boat-like conformation to Bacteroides thetaiotaomicron β-hexosaminidase (BtOG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type BtOG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of BtOG, and oxazoline (v) binds to the Asp242Asn mutant of BtOG in the 4C1 conformation.

A series of crystallographic studies on Bacteroides thetaiotaomicron β-hexosaminidase have used a variety of small molecules to define define the conformational itinerary for this family of enzymes. Substrate distortion from the 4C1 conformation found in solution to a bound 1,4B / 1S3 conformation was supported by the crystal structure of the wild-type enzyme in complex with the 7-membered azepane. [57] This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate, 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-β-D-glucopyranoside.[58] Earlier studies of the wild-type-bound thiazoline show that this intermediate is found in a 4C1conformation.[1] Subsequent studies have shown that oxazoline intermediates are bound to the mutant enzymes in this conformation.[58].


Family Firsts

First sterochemistry determination
1H-NMR studies of human O-GlcNAcase established that the β-configured hemiacetal product is formed prior to anomerisation.[2].
First catalytic nucleophile identification
This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies.[1].
First general acid/base residue identification
Studies of human O-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base.[53].
First 3-D structure
The structures of Bacteroides thetaiotaomicron O-GlcNAcase[59] and Clostridium perfringens NagJ[55].

References

  1. Macauley MS, Whitworth GE, Debowski AW, Chin D, and Vocadlo DJ. (2005). O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem. 2005;280(27):25313-22. DOI:10.1074/jbc.M413819200 | PubMed ID:15795231 [DJV2005]
  2. Greig IR, Macauley MS, Williams IH, and Vocadlo DJ. (2009). Probing synergy between two catalytic strategies in the glycoside hydrolase O-GlcNAcase using multiple linear free energy relationships. J Am Chem Soc. 2009;131(37):13415-22. DOI:10.1021/ja904506u | PubMed ID:19715310 [DJV2009]
  3. 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 | PubMed ID:16332065 [DJV2005Thio]
  4. Stubbs KA, Zhang N, and Vocadlo DJ. (2006). A divergent synthesis of 2-acyl derivatives of PUGNAc yields selective inhibitors of O-GlcNAcase. Org Biomol Chem. 2006;4(5):839-45. DOI:10.1039/b516273d | PubMed ID:16493467 [Stubbs06]
  5. Dorfmueller HC, Borodkin VS, Schimpl M, Shepherd SM, Shpiro NA, and van Aalten DM. (2006). GlcNAcstatin: a picomolar, selective O-GlcNAcase inhibitor that modulates intracellular O-glcNAcylation levels. J Am Chem Soc. 2006;128(51):16484-5. DOI:10.1021/ja066743n | PubMed ID:17177381 [Dorf2006]
  6. Dorfmueller HC, Borodkin VS, Schimpl M, Zheng X, Kime R, Read KD, and van Aalten DM. (2010). Cell-penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases. Chem Biol. 2010;17(11):1250-5. DOI:10.1016/j.chembiol.2010.09.014 | PubMed ID:21095575 [Dorf2010]
  7. Whitworth GE, Macauley MS, Stubbs KA, Dennis RJ, Taylor EJ, Davies GJ, Greig IR, and Vocadlo DJ. (2007). Analysis of PUGNAc and NAG-thiazoline as transition state analogues for human O-GlcNAcase: mechanistic and structural insights into inhibitor selectivity and transition state poise. J Am Chem Soc. 2007;129(3):635-44. DOI:10.1021/ja065697o | PubMed ID:17227027 [Whitworth2007]
  8. Mader MM and Bartlett PA. (1997). Binding Energy and Catalysis: The Implications for Transition-State Analogs and Catalytic Antibodies. Chem Rev. 1997;97(5):1281-1302. DOI:10.1021/cr960435y | PubMed ID:11851452 [Bartlett1997]
  9. He Y, Macauley MS, Stubbs KA, Vocadlo DJ, and Davies GJ. (2010). Visualizing the reaction coordinate of an O-GlcNAc hydrolase. J Am Chem Soc. 2010;132(6):1807-9. DOI:10.1021/ja9086769 | PubMed ID:20067256 [DJV2010]
  10. Macauley MS and Vocadlo DJ. (2009). Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr Res. 2009;344(9):1079-84. DOI:10.1016/j.carres.2009.04.017 | PubMed ID:19423084 [DJV2009Trunc]

  11. Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants. Biochemistry. 2006 Mar 21;45(11):3835-44.

    [DJV2006]

    Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out: DOI:10.1021/bi052370b PMID:16533067

  12. Ficko-Blean E and Boraston AB. (2005). Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005;61(Pt 9):834-6. DOI:10.1107/S1744309105024012 | PubMed ID:16511172 [ABB2005]
  13. Rao FV, Dorfmueller HC, Villa F, Allwood M, Eggleston IM, and van Aalten DM. (2006). Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis. EMBO J. 2006;25(7):1569-78. DOI:10.1038/sj.emboj.7601026 | PubMed ID:16541109 [DvA2006]
  14. Schimpl M, Schüttelkopf AW, Borodkin VS, and van Aalten DM. (2010). Human OGA binds substrates in a conserved peptide recognition groove. Biochem J. 2010;432(1):1-7. DOI:10.1042/BJ20101338 | PubMed ID:20863279 [DvA2006]
  15. Marcelo F, He Y, Yuzwa SA, Nieto L, Jiménez-Barbero J, Sollogoub M, Vocadlo DJ, Davies GD, and Blériot Y. (2009). Molecular basis for inhibition of GH84 glycoside hydrolases by substituted azepanes: conformational flexibility enables probing of substrate distortion. J Am Chem Soc. 2009;131(15):5390-2. DOI:10.1021/ja809776r | PubMed ID:19331390 [Ble2009]
  16. He Y, Macauley MS, Stubbs KA, Vocadlo DJ, and Davies GJ. (2010). Visualizing the reaction coordinate of an O-GlcNAc hydrolase. J Am Chem Soc. 2010;132(6):1807-9. DOI:10.1021/ja9086769 | PubMed ID:20067256 [GJD2010]
  17. Dennis RJ, Taylor EJ, Macauley MS, Stubbs KA, Turkenburg JP, Hart SJ, Black GN, Vocadlo DJ, and Davies GJ. (2006). Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity. Nat Struct Mol Biol. 2006;13(4):365-71. DOI:10.1038/nsmb1079 | PubMed ID:16565725 [GJD2006]
  18. Comfort DA, Bobrov KS, Ivanen DR, Shabalin KA, Harris JM, Kulminskaya AA, Brumer H, and Kelly RM. (2007). Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases. Biochemistry. 2007;46(11):3319-30. DOI:10.1021/bi061521n | PubMed ID:17323919 [Comfort2007]
  19. He S and Withers SG. (1997). Assignment of sweet almond beta-glucosidase as a family 1 glycosidase and identification of its active site nucleophile. J Biol Chem. 1997;272(40):24864-7. DOI:10.1074/jbc.272.40.24864 | PubMed ID:9312086 [He1999]
  20. [3]

  21. Sinnott, M.L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90, 1171-1202. DOI: 10.1021/cr00105a006

    [MikesClassic]

All Medline abstracts: PubMed

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