CAZypedia needs your help!
We have many unassigned pages in need of Authors and Responsible Curators. See a page that's out-of-date and just needs a touch-up? - You are also welcome to become a CAZypedian. Here's how.
Scientists at all career stages, including students, are welcome to contribute.
Learn more about CAZypedia's misson here and in this article.
Totally new to the CAZy classification? Read this first.

Glycoside Hydrolase Family 4

From CAZypedia
(Redirected from GH4)
Jump to navigation Jump to search
Approve icon-50px.png

This page has been approved by the Responsible Curator as essentially complete. CAZypedia is a living document, so further improvement of this page is still possible. If you would like to suggest an addition or correction, please contact the page's Responsible Curator directly by e-mail.

Glycoside Hydrolase Family 4
Clan none
Mechanism NAD-dependent hydrolysis
Active site residues not known
CAZy DB link

Substrate specificities

The majority of the glycoside hydrolases of this family are of bacterial origin, but they are also found in the archaeal taxa [1]. Unlike other families of glycosidases, some of the enzymes in Family 4 possess distinct substrate specificities from each other[1, 2]. This family contains α-glucosidases, α-galactosidases, α-glucuronidases, 6-phospho-α-glucosidases, and 6-phospho-β-glucosidases. Similar to GH1, some enzymes prefer phosphorylated substrates over non-phosphorylated substrates [2, 3]. Members of this family include both α- and β-glycosidases. The ability of a single enzyme to hydrolyze natural substrates of different anomeric configurations was first discovered in family GH4, and they were the first glycosidases that exhibited an absolute requirement for NAD+ and a divalent metal ion and in some instances reducing environments for catalytic activity [4, 5, 6, 7, 8, 9, 10, 11]. The cofactors are proposed to play key mechanistic roles in the atypical glycosidase mechanism. Unlike GH4, metal ions are required by some glycosidases for structural integrity [12, 13] or proper configuration of an active enzyme [14, 15, 16, 17]. Meanwhile, GH4 is the first family of glycoside hydrolases reported to require a NAD+ cofactor for catalytic activity [4, 5, 6, 7, 8, 10, 11, 18, 19, 20, 21, 22]. Subsequently, enzymes of families GH109, GH177, GH179, and GH188 has been shown to also require a NAD+ cofactor. The hydrolysis of thioglycosides with activated leaving groups has been documented in GH84 [23], GH1 [24, 25, 26, 27, 28, 29]. However, GH4 is currently the only family that has been shown to catalyze the hydrolysis of unactivated thioglycoside substrates [30]. The remarkable substrate specificities are all feasible due to the β-elimination mechanism discussed below.

Kinetics and Mechanism

GH4 enzymes have been shown to be retaining glycosidases [31, 32, 33, 34]. However, the enzymes do not utilize the classical Koshland double-displacement mechanism [35]. BglT (6-phospho-β-glucosidase) from Thermotoga maritima [36] and GlvA (6-phospho-α-glucosidase) from Bacillus subtilis [32] are two of the most thoroughly studied enzymes of this family, and both are proposed to utilize an NAD-dependent hydrolysis mechanism that proceeds through oxidation-elimination-addition-reduction steps via anionic transition states. Exchange of the substrate C2 proton with solvent deuterium during the enzyme-catalyzed reaction was the first indication of a C2-H2 bond cleavage in the substrate, which is inconsistent with the double-displacement mechanism [31, 33]. This fact coupled with the proximity of the NAD+ cofactor to C3 of the glucose 6-phosphate product and the high structural similarity to lactate/malate dehydrogenases as garnered from the x-ray crystallographic data [9, 31, 33, 34], led to the following proposed mechanism [31, 33]: 1) C3 hydride abstraction via the reduction of NAD+ cofactor to NADH and consequent oxidation of the C3 hydroxyl group; 2) adjacent to the ketone functionality, the C2 proton is activated to deprotonation by a near-by catalytic base residue; 3) cleavage of the C1-O1 linkage occurs via an α,β-elimination mechanism, producing an α,β-unsaturated intermediate; 5) a 1,4-Michael-like addition of a water molecule at C1 takes place; and 6) reduction of the C3 carbonyl functionality by the "on-board" NADH generates the final hydrolysis product.

Primary kinetic isotope effects and the determination of Brønsted relationships revealed that both the C3 hydride and C2 proton abstractions are partially rate-limiting and that cleavage of the C1-O1 linkage is not rate-limiting [32, 33, 36]. This led to the proposed E1cb mechanism for both BglT and GlvA [32, 36].

Catalytic Residues

The proposed catalytic residues were mostly derived from x-ray crystallographic data and pH-dependent activity profiles obtained for BglT and GlvA [31, 32, 33, 36]. A Tyr residue that is conserved in all 6-phospho-α- and 6-phospho-β-glycosidases was found to be approximately 4 Å away from C2 of the reaction product, which can potentially act as a general base [31, 32, 33, 34, 36]. The pH-dependent activity profiles were that of double ionization curves with pHopt at approximately 8 [32, 36]. Two pKa values of approximately 7 and 9 were determined [32, 36]. The pKa value of approximately 7 was proposed to correspond to that of the Tyr catalytic base, since this residue would need to be deprotonated for enzyme activity. The position of the Tyr in the enzyme active site was used as a possible explanation for the relatively low pKa compared to that of a free Tyr (pKa = 10). In the case of BglT, the Tyr residue is located at a distance of 3.4 Å from the glycosidic oxygen, making it ideal to assume a second role in providing general acid catalysis to the departing oxygen as well as C2 deprotonation [31, 36]. The pKa value of approximately 9 was proposed to correspond to the ionization of the conserved Arg residue(s) that were found to be within hydrogen bonding distance of the phosphate group of the substrate in these two 6-phospho-glycosidases [31, 34, 36]. Again, the normal pKa of 12 for Arg could be lowered to 9, and this matches the expectation that protonated Arg residue(s) would be needed to form electrostatic interactions with the substrate phosphate moiety. Two other conserved residues, Cys and His, were shown to be responsible for chelating the metal ion in BglT and GlvA. In the AglA structure, an Asp residue occupies the same position as the catalytic Tyr base, so the Asp is proposed to take on this role in AglA and possibly other GH4 enzymes that prefer non-phosphorylated substrates.

Three-dimensional structures

Crystallographic data is available for a number of GH4 enzymes, including α- and β-members as well as those enzymes specific for phosphorylated and non-phosphorylated substrates. The x-ray crystal structures currently available for this family are those of the Thermotoga maritima α-glucosidase AglA (PDB 1OBB) [9], the T. maritima α-glucosidase Agu4B (PDB 1VJT) [37], the Bacillus subtilis 6-phospo-α-glucosidase GlvA (PDB 1U8X) [34], and two 6-phospho-β-glucosidases: one from Geobacillus stearothermophilus (PDB 1S6Y) [37] and other BglT from T. maritima (PDB 1UP6) [31, 33]. Each enzyme possesses the characteristic dinucleotide-binding Rossman fold [38]. The crystal structures of Agu4B (PDB 1VJT) and the 6-phospho-β-glucosidase (PDB 1SY6) from do not provide useful information about the active sites. No ligands are bound in the structure of the 6-phospho-β-glucosidase from G. stearothermophilus. In the Agu4B structure, only the NAD+ cofactor is found at the enzyme active site, but no data for the nicotinamide ring is provided, possibly because it is not well-defined in the crystal structure. The AglA structure is that of an inactive enzyme with no divalent metal ion. The GH4 enzymes show some structural similarities to lactate/malate dehydrogenases as well as dehydratases [9, 31, 33, 34, 37].

Family Firsts

First stereochemistry determination
Thermotoga maritima BglT (6-phospho-β-glucosidase) via NMR and HPLC analysis of methyl-glycoside product [33]
First catalytic residue identification
Thermotoga maritima BglT (6-phospho-β-glucosidase) via x-ray crystal structure [33]
First 3-D structural determination
Thermotoga maritima AlgA (α-glucosidase) (inactive enzyme) [9]
Thermotoga maritima BglT (6-phospho-β-glucosidase) (active enzyme with all cofactors present) [31, 33]
First GH4 enzyme shown to hydrolyze both α- and β-substrates
Fusobacterium mortiferum MalH (6-phosph-α-glucosidase) [19]
First β-glycosidase
Escherichia coli CelF (6-phospho-β-glucosidase) [22]


  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 | PubMed ID:18838391 [1]
  2. Henrissat B and Davies G. (1997). Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol. 1997;7(5):637-44. DOI:10.1016/s0959-440x(97)80072-3 | PubMed ID:9345621 [2]
  3. Henrissat B and Bairoch A. (1996). Updating the sequence-based classification of glycosyl hydrolases. Biochem J. 1996;316 ( Pt 2)(Pt 2):695-6. DOI:10.1042/bj3160695 | PubMed ID:8687420 [3]
  4. Raasch C, Streit W, Schanzer J, Bibel M, Gosslar U, and Liebl W. (2000). Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase. Extremophiles. 2000;4(4):189-200. DOI:10.1007/pl00010711 | PubMed ID:10972187 [4]
  5. Thompson J, Pikis A, Ruvinov SB, Henrissat B, Yamamoto H, and Sekiguchi J. (1998). The gene glvA of Bacillus subtilis 168 encodes a metal-requiring, NAD(H)-dependent 6-phospho-alpha-glucosidase. Assignment to family 4 of the glycosylhydrolase superfamily. J Biol Chem. 1998;273(42):27347-56. DOI:10.1074/jbc.273.42.27347 | PubMed ID:9765262 [5]
  6. Thompson J, Gentry-Weeks CR, Nguyen NY, Folk JE, and Robrish SA. (1995). Purification from Fusobacterium mortiferum ATCC 25557 of a 6-phosphoryl-O-alpha-D-glucopyranosyl:6-phosphoglucohydrolase that hydrolyzes maltose 6-phosphate and related phospho-alpha-D-glucosides. J Bacteriol. 1995;177(9):2505-12. DOI:10.1128/jb.177.9.2505-2512.1995 | PubMed ID:7730284 [6]
  7. Bouma CL, Reizer J, Reizer A, Robrish SA, and Thompson J. (1997). 6-phospho-alpha-D-glucosidase from Fusobacterium mortiferum: cloning, expression, and assignment to family 4 of the glycosylhydrolases. J Bacteriol. 1997;179(13):4129-37. DOI:10.1128/jb.179.13.4129-4137.1997 | PubMed ID:9209025 [7]
  8. Thompson J, Robrish SA, Immel S, Lichtenthaler FW, Hall BG, and Pikis A. (2001). Metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Participation and properties of sucrose-6-phosphate hydrolase and phospho-alpha-glucosidase. J Biol Chem. 2001;276(40):37415-25. DOI:10.1074/jbc.M106504200 | PubMed ID:11473129 [8]
  9. Lodge JA, Maier T, Liebl W, Hoffmann V, and Sträter N. (2003). Crystal structure of Thermotoga maritima alpha-glucosidase AglA defines a new clan of NAD+-dependent glycosidases. J Biol Chem. 2003;278(21):19151-8. DOI:10.1074/jbc.M211626200 | PubMed ID:12588867 [9]
  10. Burstein C and Kepes A. (1971). The alpha-galactosidase from Escherichia coli K12. Biochim Biophys Acta. 1971;230(1):52-63. DOI:10.1016/0304-4165(71)90053-5 | PubMed ID:5543331 [10]
  11. Nagao Y, Nakada T, Imoto M, Shimamoto T, Sakai S, Tsuda M, and Tsuchiya T. (1988). Purification and analysis of the structure of alpha-galactosidase from Escherichia coli. Biochem Biophys Res Commun. 1988;151(1):236-41. DOI:10.1016/0006-291x(88)90584-0 | PubMed ID:2831880 [11]
  12. Nazmi AR, Reinisch T, and Hinz HJ. (2006). Ca-binding to Bacillus licheniformis alpha-amylase (BLA). Arch Biochem Biophys. 2006;453(1):18-25. DOI:10.1016/ | PubMed ID:16712774 [12]
  13. Vihinen M and Mäntsälä P. (1989). Microbial amylolytic enzymes. Crit Rev Biochem Mol Biol. 1989;24(4):329-418. DOI:10.3109/10409238909082556 | PubMed ID:2548811 [13]
  14. Tenu JP, Viratelle OM, and Yon J. (1972). Kinetic study of the activation process of -galactosidase from Escherichia coli by Mg 2+ . Eur J Biochem. 1972;26(1):112-8. DOI:10.1111/j.1432-1033.1972.tb01746.x | PubMed ID:4625429 [14]
  15. Case GS, Sinnott ML, and Tenu JP. (1973). The role of magnesium ions in beta-galactosidase hydrolyses. Studies on charge and shape of the beta-galactopyranosyl binding site. Biochem J. 1973;133(1):99-104. DOI:10.1042/bj1330099 | PubMed ID:4721625 [15]
  16. Withers SG, Jullien M, Sinnott ML, Viratelle OM, and Yon JM. (1978). Dependence upon pH of steady-state parameters for the beta-galactosidase-catalysed hydrolyses of beta-D-galactopyranosyl derivatives of different chemical types. Eur J Biochem. 1978;87(2):249-56. DOI:10.1111/j.1432-1033.1978.tb12373.x | PubMed ID:27358 [16]
  17. Viratelle OM and Yon JM. (1980). Comparison of the beta-galactosidase conformations induced by D-galactal and by magnesium ions. Biochemistry. 1980;19(18):4143-9. DOI:10.1021/bi00559a002 | PubMed ID:6774745 [17]
  18. Thompson J, Robrish SA, Pikis A, Brust A, and Lichtenthaler FW. (2001). Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae. Carbohydr Res. 2001;331(2):149-61. DOI:10.1016/s0008-6215(01)00028-3 | PubMed ID:11322729 [18]
  19. Pikis A, Immel S, Robrish SA, and Thompson J. (2002). Metabolism of sucrose and its five isomers by Fusobacterium mortiferum. Microbiology (Reading). 2002;148(Pt 3):843-852. DOI:10.1099/00221287-148-3-843 | PubMed ID:11882720 [19]
  20. Suresh C, Rus'd AA, Kitaoka M, and Hayashi K. (2002). Evidence that the putative alpha-glucosidase of Thermotoga maritima MSB8 is a pNP alpha-D-glucuronopyranoside hydrolyzing alpha-glucuronidase. FEBS Lett. 2002;517(1-3):159-62. DOI:10.1016/s0014-5793(02)02611-x | PubMed ID:12062428 [20]
  21. Schmitt R and Rotman B. (1966). Alpha-galactosidase activity in cell-free extracts of Escherichia coli. Biochem Biophys Res Commun. 1966;22(5):473-9. DOI:10.1016/0006-291x(66)90297-x | PubMed ID:5329906 [21]
  22. Thompson J, Ruvinov SB, Freedberg DI, and Hall BG. (1999). Cellobiose-6-phosphate hydrolase (CelF) of Escherichia coli: characterization and assignment to the unusual family 4 of glycosylhydrolases. J Bacteriol. 1999;181(23):7339-45. DOI:10.1128/JB.181.23.7339-7345.1999 | PubMed ID:10572139 [22]
  23. 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 [23]
  24. 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 | PubMed ID:3096349 [24]
  25. 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 | PubMed ID:9195886 [25]
  26. 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 | PubMed ID:10978344 [26]
  27. 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 | PubMed ID:8952475 [27]
  28. 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 | PubMed ID:3278958 [28]
  29. 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 | PubMed ID:1731996 [29]
  30. 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 | PubMed ID:16917793 [30]
  31. Varrot A, Yip VL, Li Y, Rajan SS, Yang X, Anderson WF, Thompson J, Withers SG, and Davies GJ. (2005). NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides. J Mol Biol. 2005;346(2):423-35. DOI:10.1016/j.jmb.2004.11.058 | PubMed ID:15670594 [31]
  32. Yip VL, Thompson J, and Withers SG. (2007). Mechanism of GlvA from Bacillus subtilis: a detailed kinetic analysis of a 6-phospho-alpha-glucosidase from glycoside hydrolase family 4. Biochemistry. 2007;46(34):9840-52. DOI:10.1021/bi700536p | PubMed ID:17676871 [32]
  33. Yip VL, Varrot A, Davies GJ, Rajan SS, Yang X, Thompson J, Anderson WF, and Withers SG. (2004). An unusual mechanism of glycoside hydrolysis involving redox and elimination steps by a family 4 beta-glycosidase from Thermotoga maritima. J Am Chem Soc. 2004;126(27):8354-5. DOI:10.1021/ja047632w | PubMed ID:15237973 [33]
  34. Rajan SS, Yang X, Collart F, Yip VL, Withers SG, Varrot A, Thompson J, Davies GJ, and Anderson WF. (2004). Novel catalytic mechanism of glycoside hydrolysis based on the structure of an NAD+/Mn2+ -dependent phospho-alpha-glucosidase from Bacillus subtilis. Structure. 2004;12(9):1619-29. DOI:10.1016/j.str.2004.06.020 | PubMed ID:15341727 [34]
  35. 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

  36. Yip VL and Withers SG. (2006). Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4. Biochemistry. 2006;45(2):571-80. DOI:10.1021/bi052054x | PubMed ID:16401086 [36]
  37. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, and Bourne PE. (2000). The Protein Data Bank. Nucleic Acids Res. 2000;28(1):235-42. DOI:10.1093/nar/28.1.235 | PubMed ID:10592235 [37]
  38. Buehner M, Ford GC, Olsen KW, Moras D, and Rossman MG. (1974). Three-dimensional structure of D-glyceraldehyde-3-phosphate dehydrogenase. J Mol Biol. 1974;90(1):25-49. DOI:10.1016/0022-2836(74)90254-x | PubMed ID:4375720 [38]

All Medline abstracts: PubMed