Difference between revisions of "Glycoside Hydrolase Family 52"

Revision as of 17:12, 3 September 2020

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Author: ^^^Julie Grondin^^^, ^^^Brian Lowrance^^^
Responsible Curator: ^^^Joel Weadge^^^

Glycoside Hydrolase Family GH52
Clan	GH-O
Mechanism	retaining
Active site residues	known
CAZy DB link
https://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 monospecific, functioning as exo-β-xylosidases (EC 3.2.1.37) that cleave the terminal xylose residues from the nonreducing end of xylooligosaccharides, such as 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 families for β-xylooligosaccharides and α-L-arabinofuranoside. 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 glycosyl 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 glycosyl hydrolase to a glycosynthase [9] has been achieved.

Kinetics and Mechanism

Retention of stereochemistry has been observed in GH52 β-xylosidases, 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= 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], in this structure the 6.5Å separation of Glu and Asp in the active site is 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 [].

The structure of GH52 from G. thermoglucosidasius consists of an N-terminal β-sandwich domain and a C-terminal (α/α)⁶ barrel domain, classifying these enzymes into the GH-O clan, similar to that noted for the GH116 family. The exo-acting mode of action of GH52's is reflected in the topology of the active site. The enzyme acts as a dimer in solution [1, 2], with interactions between monomers forming a deep pocket to enclose and distort the xylose on the non-reducing end 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 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. 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: GH52 from Geobacillus thermoglucosidasius NBRC 107763 [2].

References

All Medline abstracts: PubMed

@@ Line 40: / Line 40: @@
 [[File:Figure1_dimer.PNG|300px|thumb|right|'''Figure 1. The dimeric structure of GH52 from ''Geobacillus thermoglucosidasius'' in complex with xylobiose (orange)([{{PDBlink}}4C1P 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 <cite>Espina2014</cite>.]]
-The structure of GH52 from ''G. thermoglucosidasius'' consists of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain, classifying these enzymes into the GH-''O'' clan, similar to that noted for the GH116 family. The exo-acting mode of action of GH52 is reflected in the topology of the active site. The enzyme acts as a dimer in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers forming a deep pocket to enclose and distort the non-reducing end xylose into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site 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. This mechanism promotes strict ''exo''-&beta;-xylosidase activity while inhibiting activity on large polymers such as xylan.
+The structure of GH52 from ''G. thermoglucosidasius'' consists of an N-terminal β-sandwich domain and a C-terminal (&alpha;/&alpha;)<sup>6</sup> barrel domain, classifying these enzymes into the GH-''O'' clan, similar to that noted for the GH116 family. The exo-acting mode of action of GH52's is reflected in the topology of the active site. The enzyme acts as a dimer in solution <cite>Bravman2001, Espina2014</cite>, with interactions between monomers forming a deep pocket to enclose and distort the xylose on the non-reducing end into a high-energy <sup>4</sup>H<sub>3</sub> half-chair transition conformation, while simultaneously hindering the entry of large xylan polymers into the catalytic site <cite>Espina2014</cite>. Furthermore, the structure of the active site 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. This mechanism promotes strict ''exo''-&beta;-xylosidase activity while inhibiting activity on large polymers such as xylan.
 == Family Firsts ==