File:Gh12 2.png

2015-07-28T14:39:25Z

Gerlind Sulzenbacher:

Glycoside Hydrolase Family 12

2015-07-28T14:38:37Z

Gerlind Sulzenbacher:

{{UnderConstruction}}
* [[Author]]s: ^^^Mats Sandgren^^^ and ^^^Gerlind Sulzenbacher^^^
* [[Responsible Curator]]: ^^^Gerlind Sulzenbacher^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH12'''
|-
|'''Clan'''
|GH-C
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH12.html
|}
</div>


== Substrate specificities ==
The substrate specificities found among the [[glycoside hydrolases]] of family 12 are: ''[[endo]]''-β-1,4-glucanase (EC [{{EClink}}3.2.1.4 3.2.1.4]), xyloglucan ''[[endo]]''-hydrolase (EC [{{EClink}}3.2.1.151 3.2.1.151]), ''[[endo]]''-β-1,3-1,4-glucanase (EC [{{EClink}}3.2.1.73 3.2.1.73]). Xyloglucan ''[[endo]]''-transglycosylase (XET, EC [{{EClink}}2.4.1.207 2.4.1.207]) activity has been observed in a single fungal GH12 member (GenBank [http://www.ncbi.nlm.nih.gov/protein/AAN89225.1 AAN89225.1]) using a XET-specific screen <cite>Nielsen2002</cite>, although this may represent a side activity of a predominant xyloglucan ''[[endo]]''-hydrolase <cite>Gilbert2008 Eklof2010</cite>.

== Kinetics and Mechanism ==
GH12 enzymes are [[retaining]] enzymes, as first shown by NMR studies <cite>Schou1993</cite> on endoglucanase 3 from ''Humicola insolens'', and is believed to follow a classical [[Koshland double-displacement mechanism]] in which a glycosyl-enzyme intermediate is formed and subsequently this intermediate is hydrolysed via oxocarbenium-ion [[transition state]]s. No detailed studies involving both steady state and pre-steady state kinetic have yet been reported for GH12.

== Catalytic Residues ==
[[File:gh12_1.png|thumb|300px|right]]
The [[catalytic nucleophile]] and the [[general acid/base]] catalyst of GH12 enzymes was initialy predicted by sequence homology to the xylanase members of [[GH11]], a glycoside hydrolase family where the [[catalytic nucleophile]] was first identified in the ''Bacillus circulans'' [[endo]]-xylanase through trapping of the 2-deoxy-2-fluoroxylobiosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies <cite>Miao1994</cite>. [[GH11]] and GH12 together form [[clan]] GH-C. The prediction of the [[catalytic nucleophile]] and the [[general acid/base]] of GH family 12 enzymes was later confirmed to be correct when the first three dimensional structure of a GH family 12 enzyme was determined, that of ''Streptomyces Lividans'' CelB <cite>Sulzenbacher1997</cite>. The [[catalytic nucleophile]] was subsequentialy confirmed in ''Streptomyces Lividans'' CelB to be Glu 120 by using the same labeling strategy used for detecting the [[catalytic nucleophile]] of GH family 11 <cite>Zechel1998</cite>.

== Three-dimensional structures ==
Enzymes from family GH12 adopt a “jelly-roll” fold with two twisted, mostly antiparallel β-sheets stacking on top of each other and enclosing a long substrate-binding groove located on the concave face of the sheet. A single α-helix packs against the convex surface of the outer β-sheet. GH12 members of bacterial origin feature an additional two-stranded β-sheet, stabilized by a disulphide bridge, located at the rim of the substrate binding groove and providing an additional substrate binding platform as compared to the eukaryotic counterparts. The first structural template provided for family GH12 was that of ''Streptomyces lividans'' CelB2 in the apo-form <cite>Sulzenbacher1997</cite> and in the form of a glycosyl-enzyme intermediate <cite>Sulzenbacher1997</cite>. This first structure confirmed previous predictions, based on hydrophobic cluster analysis <cite>Torronen1993</cite>, that GH12 enzymes would display significant structural similarity with family [[GH11]] xylanases and gave rise to the creation of clan GH-C, which so far comprises only the two above families. The first structure of a fungal GH12 enzyme was provided shortly after for ''Trichoderma reesei'' (formerly ''Hypocrea jecorina'') Cel12A <cite>Sandgren2001</cite>. To date structural models, both in the apo-form and in complex with oligosaccharides, are available for sixteen family members. From the different complex structures it can be seen that the substrate-binding groove of family GH12 enzymes harbours six binding sites, from -4 to +2, lined by numerous aromatic residues providing staking platforms for the individual pyranose units. This is consistent with the endoglucanase activity of these enzymes, exhibiting low catalytic efficiency towards short chain substrates <cite>Karlsson2002</cite>. The [[catalytic nucleophile]] Glu120 (S. lividans CelB2 numbering) and the [[ general acid/base]] Glu203 are in close proximity of an invariant aspartate, which might have an important role in catalysis and the constellation of this catalytic triad is reminiscent of the catalytic centre of family GH7 enzymes. Like in family [[GH7]] enzymes the acid/base residues of family GH12 members protonate syn to the pyranoside 0-5-C-1 bond.

For xyloglucan specific enzymes of the family, structural and functional studies of xyloglucanase XG12 from ''Bacillus licheniformis'' <cite>Gloster2007</cite> and the xyloglucan specific endo-β-1,4-glucanase XEG1 from ''Aspergillus aculeatus KSM 50'' <cite>Yoshizawa2012</cite> have shown that α-1,6-xylose substitutions can be tolerated at subsites -3, -2, +1 and +2. Certain GH12 enzymes exhibit β-1,3-1,4-glucanase activity and the crystal structure of ''Humicola grisea'' Cel12A in complex with a mixed linkage oligosaccharide arising from a transglycosylation reaction revealed that the β-1,3-linkage can be accommodated with ease between subsites -3 and -2 <cite>Sandgren2004</cite>. Speculations had been forwarded that a long loop crossing the substrate-binding groove at its reducing end, known as the “cord” and conserved in all clan GH-C members, might undergo conformational changes upon substrate binding <cite>Torronen1994</cite>, but the available GH12 complex structures support the view that the role of this loop is to deliver residues for substrate binding.

The crystal structure of Aspergillus aculeatus XEG1 in complex with the glycoside hydrolase inhibitory protein from carrot. EDGP, reveals an original mode of inhibition, distinct from the one observed previously for the inhibition of a family [[GH11]] xylanase by a wheat protein <cite>Payan2004</cite>. In the XEG1-GHIP complex the inhibitor mimics a xyloglucan substrate and inserts two conserved arginine residues into the active site, which establish salt bridges with the carboxylate groups of the catalytic centre <cite>Yoshizawa2012</cite>).

Due to their commercial interest in bioprocessing applications, GH12 endo-β-1,4-glucanases from ''Humicola grisea'' <cite>Sandgren2003a</cite>, ''Trichoderma reesei'' <cite>Sandgren2003a,Sandgren2003ab</cite>, ''Rhodothermus marinus'' <cite>Kapoor2008</cite>, ''Pyrococcus furiosus'' <cite>Kim2012</cite> and ''Thermotoga maritima'' <cite>Cheng2012</cite> have been extensively engineered and the structural studies of the resulting variants have provided valuable insight into structural features governing enzyme activity and stability.

== Family Firsts ==
;First sterochemistry determination: ''Humicola insolens'' endoglucanase 3 by NMR <cite>Schou1993</cite>.
;First catalytic nucleophile identification: .
;First general acid/base residue identification: .
;First 3-D structure: .

== References ==
<biblio>
#Schou1993 pmid=8223652
#Sulzenbacher1997 pmid=7911679
#Miao1994 pmid=9440876
#Zechel1998 pmid=9806895
#Gilbert2008 pmid=18430603
#Eklof2010 pmid=20421457
#Sandgren2003a pmid=14627738
#Sandgren2003b pmid=12649442
#Sandgren2001 pmid=11327768
#Sandgren2004 pmid=15364577
#Torronen1993 pmid=8477842
#Toronnen1994 pmid=8013449
#Payan2004 pmid=15181003
#Yoshizawa2012 pmid=22496365
#Gloster 2007 pmid=17376777
#Kim2012 pmid=22569255
#Cheng2012 pmid=22170108
#Kapoor2018 pmid=18555809
#Karlsson 2002 pmid=12204558
#Sulzenbacher1999 pmid=10200171
#Nielsen2002 Nielsen, RI. (2002) ''Microbial xyloglucan endotransglycosylase (XET)'' Patent [{{PatentLink}}US6448056 US6448056].
</biblio>

[[Category:Glycoside Hydrolase Families|GH012]]

2010-01-13T12:36:11Z

Gerlind Sulzenbacher:

{{UnderConstruction}}
* [[Author]]: ^^^Gerlind Suzlenbacher^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH 29'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |http://www.cazy.org/fam/GH29.html
|}
</div>


== Substrate specificities ==
The [[glycoside hydrolases]] of this family are exo-acting α-fucosidases from archaeal, bacterial and eukaryotic origin. No other activities have been observed for GH29 family members. So fare the only other CAZY family containing α-fucosidases is family [[GH95]]. The human enzyme FucA1 is of medical interest because its deficiency leads to fucosidosis, an autosomal recessive lysosomal storage disease <cite>1</cite>.

== Kinetics and Mechanism ==
GH29 α-fucosidases are [[retaining]] enzymes following a [[classical Koshland double-displacement mechanism]], as first proposed in 1987 for human liver α-fucosidase via burst kinetics experiments and using methanol as an alternative glycone acceptor to produce methyl-α-L-fucoside <cite>2</cite>. This has been further confirmed by <sup>1</sup>H NMR monitoring of the reaction catalyzed by a α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>, and a α-L-fucosidase from the marine mollusc ''Pecten maximus''<cite>4</cite>, as well as by COSY and <sup>1</sup>H-<sup>13</sup>C NMR spectroscopy analysis of the interglycosidic linkage of disaccharides formed by the transglycosylation action of ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc <cite>5</cite>. [[GH95]] α-fucosidases, in contrast, operate with inversion of the anomeric configuration.

== Catalytic Residues ==
The [[catalytic nucleophile]] in GH29 was first identified in the ''Sulfolobus solfataricus'' α-L-fucosidase, Ssα-fuc, as Asp242 in the sequence VYF<u>'''D'''</u>WWI via chemical rescue of an inactive mutant with sodium azide <cite>6</cite>. Concomitantly the [[catalytic nucleophile]] of ''Thermotoga maritima'' α-L-fucosidase, Tmα-fuc, was confirmed to be Asp224 in the sequence LWN<u>'''D'''</u>MGW through trapping of the 2-deoxy-2-fluorofucosyl-enzyme [[intermediate]] and subsequent peptide mapping via LC-MS/MS technologies, as well as by chemical rescue of an inactive mutant <cite>7</cite>. The trapping of the 2-deoxy-2-fluorofucosyl-enzyme intermediate in Tmα-fuc was corroborated by crystallographic studies <cite>8</cite>. The [[catalytic nucleophile]] of the human enzyme FucA1 has recently been identified as being Asp225 <cite>9</cite>.

Whereas the [[catalytic nucleophile]] in GH29 has been shown to be a conserved aspartate residue, the identity of the [[general acid/base]] is still controversial. Structural and mutagenesis studies of Tmα-fuc provided strong evidence for the variant Glu266 being the [[general acid/base]] <cite>8</cite>. In the crystal structure the carboxyl function of this residue is 5.5 Å apart from that of the [[catalytic nucleophile]] Asp224, a distance commonly observed in retaining glycosidases proceeding via a [[classical Koshland double-displacement mechanism]]. Although multiple sequence alignments show that Glu266 is not conserved within GH29, the residue is structurally conserved in two 3-D structures of α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', recently deposited in the [http://www.pdb.org/ Protein Data Bank] (accession numbers 3eyp and 3gza). Studies of Ssα-fuc demonstrated that mutation of the Glu residue corresponding in sequence to Tmα-fuc Glu266 scarcely impaired the catalytic activity of the enzyme, whereas the E58G mutant yielded a 4000-fold reduction of ''k<sub>cat</sub>/K<sub>M</sub>'' and could be chemically rescued <cite>10</cite>. In the crystal structure of Tmα-fuc in complex with fucose <cite>8</cite>, the residue corresponding to Ssα-fuc Glu58, Glu66, is found 7.5 Å distant form the [[catalytic nucleophile]] Asp224 and hydrogen bond to the C-3 hydroxyl group of fucose, which altogether makes this residue an unlikely candidate for the function of the [[general acid/base]]. A recent study of the human α-L-fucosidase FucA1, carefully done combining bioinformatics, structural modelling, mutagenesis, chemical rescue with azide and <sup>1</sup>H NMR spectral analysis, identified Glu289 as the [[general acid/base]] <cite>9</cite>. The equivalent residue in Tmα-fuc, Glu281, as inferred from sequence alignment of FucA1 and Tmα-fuc, points to the interior of the (β/α)<sub>8</sub> barrel and lies about 15 Å apart form the catalytic centre.

Altogether it appears that family GH29 is a quite exceptional CAZy family in that multiple sequence alignments do not allow an unambiguous assignment of the [[general acid/base]].

== Three-dimensional structures ==
Very few structures are available for GH29 enzyme. The first crystal structure being solved is the one for the α-L-fucosidase from ''T. maritima'', Tmα-fuc. The simultaneous solution of the structures of an enzyme-product complex and of a glycosyl-enzyme intermediate allowed the unambiguous identification of the [[general acid/base]] <cite>8</cite>, as described above. Tmα-fuc assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)<sub>8</sub>-like domain and a C-terminal β-sandwich domain. The two key active site residues are located at the C-terminal ends of strands β-strands 4 (nucleophile) and 6 (acid/base).
Crystallization experiments for the ''S. solfataricus'' α-L-fucosidase, Ssα-fuc, were not very fruitful, but a small angle scattering study has been reported <cite>11</cite>, which suggests a nonameric assembly of the enzyme in solution. Two crystal structures, arising for Strucural Genomics initiatives, have been deposited in the [http://www.pdb.org/ Protein Data Bank] for α-L-fucosidases from ''Bacteroides thetaiotaomicron VPI-5482'', with accession numbers 3eyp and 3gza.
The catalytic domain of Tmα-fuc does not adopt the canonical TIM-barrel (β/α)<sub>8</sub> fold, as it lacks helices α5 and α6. Helix α5 is missing as well in the structure of one of the ''B. thetaiotaomicron VPI-5482'' α-L-fucosidases, BT3798 (3gza), whereas α-L-fucosidase BT2192 (3epy) from the same organism adopts the canonical TIM-barrel fold. The three structures differ furthermore by the insertion/deletion of a considerable number of additional α-helices, 3<sub>10</sub> helices, and extended surface loop regions. The closest structural homologues of GH29 enzymes within the CAZy classification can be found in families [[GH13]] and [[GH27]].

== Fucosynthases ==
Transglycosylation activity had been observed in 1987 for human liver α-fucosidase <cite>2</cite>. The first successful transformation of an α-fucosidase into an α-transfucosidase by directed evolution has been reported for ''Thermotoga maritima'' α-fucosidase <cite>12</cite>. α-fucosidases mutated in the [[catalytic nucleophile]] from both ''Sulfolobus solfataricus'' and ''Thermotoga maritima'' could be successfully transformed into fucosynthases by the use of β-L-fucopyranosyl azide as donor substrate <cite>13</cite>

== Family Firsts ==
;First stereochemistry determination: Retention of anomeric stereochemistry suggested for human liver α-fucosidase by the formation of methyl-α-L-fucoside using methanol as an alternative glycone acceptor <cite>2</cite>. Later confirmed by <sup>1</sup>H NMR for α-L-fucosidase from ''Thermus sp.'' <cite>3</cite>.
; First [[catalytic nucleophile]] identification : ''Sulfolobus solfataricus'' α-L-fucosidase by azide rescue of an inactivated mutant <cite>6</cite>.
; First [[general acid/base]] residue identification : ''Thermotoga maritima'' α-fucosidase by X-ray structural analysis and mutagenesis <cite>8</cite>.
; First 3-D structure : ''Thermotoga maritima'' α-fucosidase, free enzyme ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL8 PDB 1hl8]), product complex ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1ODU PDB 1odu]) and glycosyl-enzyme intermediate ([http://www.rcsb.org/pdb/explore/explore.do?structureId=1HL9 PDB 1hl9]) <cite>8</cite>.

== References ==
<biblio>
#1 pmid=2894306
#2 pmid=3828350
#3 pmid=12441672
#4 pmid=12042250
#5 pmid=12569098
#6 pmid=12911294
#7 pmid=12975375
#8 pmid=14715651
#9 pmid=19072333
#10 pmid=15835922
#11 pmid=15207718
#12 pmid=17240986
#13 pmid=17240986
DOI: 10.1016/j.chembiol.2009.09.013

</biblio>

[[Category:Glycoside Hydrolase Families|GH029]]