Glycoside Hydrolase Family 52

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• Author: ^^^Julie Grondin^^^ and ^^^Brian Lowrance^^^
• Responsible Curator: ^^^Joel Weadge^^^

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Glycoside Hydrolase Family GH52
Clan	GH-O
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH52.html

Substrate specificities

The GH52 enzymes are often isolated from various mesophilic and thermophilic bacteria, which has led to a demonstrated high thermostability within this family. The enzymes are generally monospecific, functioning as exo-β-xylosidases (EC 3.2.1.37) that cleave the terminal xylose residues from the non-reducing end of artificial xylosides and xylooligosaccharides (e.g., pNP-β-D-xylopyranoside [1, 2], xylobiose [2], and xylotriose [2]). Low levels of α-L-arabinofuranoside activity has also been observed within members of the GH52 family [3, 4], which is similar to the specificity noted for GH13 and GH54 for β-xylooligosaccharides and α-L-arabinofuranosides. The specificity for these substrates is likely due to similarities in orientation of hydroxyls and glycosidic bonds of the substrate within the active site [5, 6]. Under certain conditions, some enzymes in the family have also exhibited weak transglycosylation activity, a phenomenon that has also been infrequently observed in other glycoside hydrolases [7]. The plasticity of the active site of some GH52 members has been further explored through site-directed mutagenesis, where introduction of xylanase activity [8] and transition from a glycoside hydrolase to a glycosynthase [9] has been achieved.

Kinetics and Mechanism

Retention of stereochemistry has been observed in GH52 β-xylosidases, which is characteristic of a classical Koshland double-displacement mechanism [10]. This was first determined by Bravmen and coworkers using ¹H-NMR to analyze the breakdown products of pNP-β-D-xylopyranoside by XynB2, a β-xylosidase from Bacillus stearothermophilus T-6 [1]. Further detailed analysis within this family was published in 2003 on the B. stearothermophilus XynB2 enzyme, which contained pH dependence studies (enzymatic catalysis is dependent on ionizable residues E335 and D495, with free enzyme experimental pKa values of 4.2 and 7.3, respectively) and kinetic analyses (pNP-xylobiose k_cat/K_M of 140 s^-1mM^-1; xylobiose and xylotriose K_M values of 17.1x10⁴ M^-1 and 9.6x10⁴ M-¹, respectively) [4].

Catalytic Residues

Site-directed mutagenesis, chemical rescue, and kinetic profiling of XynB2 from B. stearothermophilus T-6 identified E335 as the catalytic nucleophile and D495 as the general acid/base [1, 11]. The catalytic nucleophile (E335) is conserved within the WVVNEGEY motif, which is found approximately 150 residues up-stream from the EITTYDSLD motif containing the general acid/base (D495). These results were further confirmed following the structural analysis of a GH52 from Geobacillus thermoglucosidasius [2], where in this structure the 6.5 Å separation of Glu and Asp in the active site was typical of retaining enzymes.

Three-dimensional structures

Figure 1. The dimeric structure of GH52 from Geobacillus thermoglucosidasius in complex with xylobiose (orange)(PDB ID 4C1P). The active site is enclosed by residues from both monomers, restricting this enzyme to exo-hydrolysis via steric hindrance of the catalytic site. Figure from [2].

Representative structures of GH52 glycoside hydrolases have been solved, XynB2 from B. stearothermophilus T-6 (PDB ID 4RHH) and GT2_24_00240 from G. thermoglucosidasius (PDB ID 4C1P; PDB ID 4C1O). These enzymes have folds comprised of an N-terminal β-sandwich domain and a C-terminal (α/α)⁶ barrel domain (Figure 1) that has led to their classification into Clan GH-O, together with GH116. The exo-acting mode-of-action of GH52's is reflected in the topology of the active site. The enzymes act as dimers in solution [1, 2], with interactions between monomers of the GH52 from G. thermoglucosidasius (PDB ID 4C1P) forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy ⁴H₃ half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site [2]. Furthermore, the structure of the active site also allosterically inhibits access to negative subsites beyond the -1 site. This permits interaction with only a single xylosyl residue in the negative subsites and thus hydrolysis yields a lone xylose molecule. In summary, this mechanism promotes strict exo-β-xylosidase activity, while inhibiting activity on large polymers, such as xylan.

Family Firsts

First stereochemistry determination

XynB2 from Bacillus stearothermophilus T-6 by ¹H-NMR for the hydrolysis of pNP-β-D-xylopyranoside [1].

First catalytic nucleophile identification

XynB2 from Bacillus stearothermophilus T-6 by site-directed mutagenesis and chemical rescue [12].

First general acid/base residue identification

XynB2 from Bacillus stearothermophilus T-6 by site-directed mutagenesis, chemical rescue, and pH profiling [12].

First 3-D structure

GT2_24_00240 from Geobacillus thermoglucosidasius NBRC 107763 [2].

References

1. Bravman T, Zolotnitsky G, Shulami S, Belakhov V, Solomon D, Baasov T, Shoham G, and Shoham Y. (2001). Stereochemistry of family 52 glycosyl hydrolases: a beta-xylosidase from Bacillus stearothermophilus T-6 is a retaining enzyme. FEBS Lett. 2001;495(1-2):39-43. DOI:10.1016/s0014-5793(01)02360-2 |
2. Espina G, Eley K, Pompidor G, Schneider TR, Crennell SJ, and Danson MJ. (2014). A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds. Acta Crystallogr D Biol Crystallogr. 2014;70(Pt 5):1366-74. DOI:10.1107/S1399004714002788 |
4. Bravman T, Zolotnitsky G, Belakhov V, Shoham G, Henrissat B, Baasov T, and Shoham Y. (2003). Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus. Biochemistry. 2003;42(35):10528-36. DOI:10.1021/bi034505o |
5. Lee TH, Lim PO, and Lee YE. (2007). Cloning, characterization, and expression of xylanase A gene from Paenibacillus sp. DG-22 in Escherichia coli. J Microbiol Biotechnol. 2007;17(1):29-36. | Google Books | Open Library
6. Utt EA, Eddy CK, Keshav KF, and Ingram LO. (1991). Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with beta-D-xylosidase and alpha-L-arabinofuranosidase activities. Appl Environ Microbiol. 1991;57(4):1227-34. DOI:10.1128/aem.57.4.1227-1234.1991 |
7. Romero-Téllez S, Lluch JM, González-Lafont À, and Masgrau L. (2019). Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study. Front Chem. 2019;7:200. DOI:10.3389/fchem.2019.00200 |
8. Huang Z, Liu X, Zhang S, and Liu Z. (2014). GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site‑directed mutagenesis of Tyr509. J Ind Microbiol Biotechnol. 2014;41(1):65-74. DOI:10.1007/s10295-013-1351-x |
9. Dann R, Lansky S, Lavid N, Zehavi A, Belakhov V, Baasov T, Dvir H, Manjasetty B, Belrhali H, Shoham Y, and Shoham G. (2014). Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase from Geobacillus stearothermophilus T6. Acta Crystallogr F Struct Biol Commun. 2014;70(Pt 12):1675-82. DOI:10.1107/S2053230X14023887 |

10. 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

11. Bravman T, Belakhov V, Solomon D, Shoham G, Henrissat B, Baasov T, and Shoham Y. (2003). Identification of the catalytic residues in family 52 glycoside hydrolase, a beta-xylosidase from Geobacillus stearothermophilus T-6. J Biol Chem. 2003;278(29):26742-9. DOI:10.1074/jbc.M304144200 |
13. Suzuki T, Kitagawa E, Sakakibara F, Ibata K, Usui K, and Kawai K. (2001). Cloning, expression, and characterization of a family 52 beta-xylosidase gene (xysB) of a multiple-xylanase-producing bacterium, Aeromonas caviae ME-1. Biosci Biotechnol Biochem. 2001;65(3):487-94. DOI:10.1271/bbb.65.487 |