{{CuratorApproved}}
* [[Author]]: ^^^Gideon Davies^^^
* [[Responsible Curator]]: ^^^Gideon Davies^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH5'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH5.html
|}
</div>


== Substrate specificities ==
GH5 is one of the largest of all CAZy [[glycoside hydrolase]] families. Previously known as "cellulase family A" <cite>Henrissat1989 Gilkes1991</cite>, a variety of specificities are now known in this family, notably endoglucanase (cellulase) and endomannanase, as well as exoglucanases, exomannanases and β-glucosidase and β-mannosidase. Other activities include 1,6-galactanase, 1,3-mannanase, 1,4-xylanase, endoglycoceramidase, as well as high specificity xyloglucanases. Family GH5 enzymes are found widely distributed across Archae, bacteria and eukaryotes, notably fungi and plants. There are no known human enzymes in GH5. Following the reclassification of a number of GH5 members into [[GH30]] <cite>StJohn2010</cite>, a GH5 subfamily classification has been presented that delineates members into a number of monospecific and polyspecific clades <cite>Aspeborg2012</cite>. It should be noted that enzymes specifically targeting xylans are exclusively arabinoxylanases, and are found in subfamilies GH_21 and GH_34. A similar subfamily classification was previously devised for [[GH13]] to aid functional prediction <cite>Stam2006</cite>.

== Kinetics and Mechanism ==
Family GH5 enzymes are [[retaining]] enzymes, as first shown by NMR <cite>Barras1992</cite> and follow a [[classical Koshland double-displacement mechanism]].

== Catalytic Residues ==
GH5 enzymes use the [[classical Koshland double-displacement mechanism]] and the two catalytic residues ([[catalytic nucleophile]] and [[general acid/base]]) are known to be glutamates found at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) <cite>Henrissat1996 Jenkins1995</cite>.

== Three-dimensional structures ==
Three-dimensional structures are available for a very large number of Family GH5 enzymes, the first solved being that of the ''Clostridium thermocellum'' endoglucanase CelC <cite>Alzari1995</cite>. As members of [[Clan]] GH-A they have a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) <cite>Henrissat1996 Jenkins1995</cite>.

With so many 3D structures in this family, covering many specificities it is clearly hard to pick out notable structural papers. The ''Bacillus agaradhaerens'' Cel5A has been extensively studied, notably in the trapping of enzymatic snapshots along the reaction coordinate <cite>Davies1998</cite> but also as a testbed for glycosidase inhibitor design as crystals often diffract to atomic resolution (for example <cite>Varrot2003</cite>). The reaction coordinate work on the endoglucanases (thus working on ''gluco''-configured substrates) shows that the substrate binds in 1S3 conformation with the glycosyl enzyme [[intermediate]] in 4''C''1 chair conformation implying catalysis via a near 4''H''3 half-chair [[transition state]].

By analogy with family [[GH26]] mannnanases <cite>Ducros</cite> and family [[GH2]] β-mannosidases <cite>Tailford</cite> it would seem likely that GH5 mannanases use a different conformational itinerary to their glucosidase relatives, likely via a 1''S''5-O''S''2 glycosylation pathway and thus ''via'' a ''B''2,5 (near) transition-state although direct evidence in this family is limited <cite>Vincent</cite>. An interesting dissection of mannan-degrading enzyme systems has been provided by work in the Gilbert group on the diverse GH5 and [[GH26]] mannanases in ''Cellvibrio japonicus''(see for example <cite>Hogg,Tailford-2 Cartmell2008</cite>).

The Rhodococcal endoglycoceramidase II (EGC) in this family has found application in the chemoenzymatic synthesis of ceramide derivatives <cite>Caines2007</cite>. In 2007 the first 3-D structure of a highly specific GH5 xyloglucanase was reported <cite>Gloster2007</cite>; this enzyme makes kinetically productive interactions with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides.

== Family Firsts ==
;First sterochemistry determination: The curator believes this to be the 1H NMR stereochemical determination for EGZ from ''Erwinia chrysanthemi'' <cite>Barras1992</cite>. GH5 enzymes were also in the comprehensive Gebler study <cite>Gebler1992</cite>.
;First [[catalytic nucleophile]] identification: Trapped using the classical Withers 2-fluoro method, here with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside, reported in Wang and Withers in 1993 <cite>Wang1993</cite>.

;First [[general acid/base]] identification: Several mutagenesis papers has alluded to the importance of a conserved glutamate- one that both Dominguez <cite>Dominguez1995</cite> and Ducros <cite>Ducros1995</cite> correctly postulated as the catalytic acid when the 3-D structures were determined.

;First 3-D structure: The first 3D structures in family GH5 was an endoglucanase (cellulase) from ''Clostridium thermocellum'' reported by the Alzari in 1995 (in a paper which also reported a family GH10 xylanase structure and the similarities between them) <cite>Dominguez1995</cite>. Subsequently, Ducros and colleagues reported the ''Clostridium cellulolyticum'' Cel5A also in 1995 <cite>Ducros1995</cite>.

== References ==
<biblio>
#Jenkins1995 pmid=7729513
#Henrissat1996 pmid=8643635
#Caines2007 pmid=17329247
#Barras1992 pmid=1563515
#Wang1993 pmid=8100226
#Gebler1992 pmid=1618761
#Dominguez1995 pmid=7664125
#Ducros1995 pmid=8535787
#Davies1998 pmid=9718293
#Varrot2003 pmid=12812472
#Gloster2007 pmid=17376777
#Ducros pmid=12203498
#Tailford pmid=18408714
#Tailford-2 pmid=19441796
#Hogg pmid=12523937
#Vincent pmid=15515081
#Cartmell2008 pmid=18799462
#Aspeborg2012 pmid=22992189
#StJohn2010 pmid=20932833
#Henrissat1989 pmid=2806912
#Stam2006 pmid=17085431
#Gilkes1991 pmid=1886523
</biblio>

[[Category:Glycoside Hydrolase Families|GH005]]

2018-02-19T19:51:58Z

Harry Gilbert: /* Catalytic Residues */

{{CuratorApproved}}
* [[Author]]: ^^^Harry Gilbert^^^
* [[Responsible Curator]]: ^^^Henrik Stalbrand^^^
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH26'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH26.html
|}
</div>

== Substrate specificities ==
[[Glycoside hydrolases]] of family 26 are primarily of [[endo]]-β-1,4-mannanases, although a recent [[exo]]-acting β-mannanase has been described <cite>Cartmell2008</cite>. The family also contains enzymes that display β-1,3:1,4-glucanase <cite>Taylor2005</cite> and β-1,3-xylanase activities <cite>Araki2000</cite>. As a historical note, GH26 was one of the first glycoside hydrolase families classified by sequence analysis, and was previously known as "Cellulase Family I (eye)" prior to detailed enzymological characterization <cite>Gilkes1991</cite>.

== Kinetics and Mechanism ==
Family GH26 enzymes utlize a [[retaining]] mechanism, as shown by NMR and follow a classical [[Koshland double-displacement mechanism]]. Pre-steady state kinetics using activated substrates revealed the two phases of the reaction; the rapid initial glycosylation step (only with good leaving groups) followed by the slower deglycosylation. It should be noted that the use of substrates with a good leaving group result in a very low apparent KM, particularly with the acid-base mutant. This does not reflect tight affinity but simply that the glycosylation step (k2) is much quicker than the deglycosylation step (k3) <cite>Bolam1996</cite>.

== Catalytic Residues ==
The catalytic residues were first identified in the [[endo]]-β-1,4-mannanase CjMan26A. The [[general acid/base]] residue is the glutamate Glu320, which is separated in sequence by ~100 residues from the [[catalytic nucleophile]], Glu212. The [[catalytic nucleophile]] was identified by site-directed mutagenesis in harness with the kinetics of 2,4-dintrophenyl-β-mannobioside hydrolysis which, although very slow was associated with a dramatic decrease in KM <cite>Bolam1996</cite>. The identity of the [[catalytic nucleophile]] was also revealed through site-directed mutagenesis <cite>Bolam1996</cite> and its function was visualized by X-ray crystallography in the acid-base mutant of which the glycosyl enzyme [[intermediate]] bound to 2-deoxy-2-fluoromannose was formed <cite>Ducros2002</cite>. In [[Sequence-based classification of glycoside hydrolases|Clan]] GHA, of which GH26 is a member, the residue immediately preceding the [[general acid/base]] residue in sequence is an asparagine that makes pivotal interactions with the 2-hydroxyl of the substrate. In GH26 the equivalent amino acid is a histidine, His211 in CjMan26A, although its function is conserved; it also makes important interactions with the 2-hydroxyl of the substrate <cite>Ducros2002</cite>.

== Three-dimensional structures ==
Three-dimensional structures are available for a large number of Family GH26 enzymes, the first solved being that of the ''Cellvibrio japonicus'' (previously called various names in the genus ''Pseudomonas'') mannanase CjMan26A <cite>#6</cite>. As members of Clan GHA they have a classical (α/β)8 TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile). The crystal structure of two ''C. japonicus'' mannanases in complex with activated substrates with the acid base mutant <cite>#5</cite>, or substrates that are very slowly hydrolyzed in the wild type enzyme <cite>Cartmell2008</cite>, show that catalysis by this class of enzyme proceeds via a Boat2,5 (''B''2,5) [[transition state]], whereas the GH26 β-1,3:1,4-glucanase [[transition state]] adopts a half-chair 4H3 conformation <cite>#7</cite>. The chemical rationale for the different [[transition state]]s adopted by β-mannanases and glucanases is discussed by Davies and colleagues in these publications and elsewhere <cite>#8</cite>. The crystal structures have also revealed the mechanism of substrate recognition in subsites distal to -1 <cite>#9 #10</cite>.

== Family Firsts ==
;First sterochemistry determination: ''Cellvibrio japonicus'' CjMan26A by NMR <cite>#4</cite>.
;First [[catalytic nucleophile]] identification: ''Cellvibrio japonicus'' CjMan26A initially by mutagenesis and sequence conservation <cite>#4</cite> and later by X-ray crystallography <cite>#5</cite>.
;First [[general acid/base]] residue identification: ''Cellvibrio japonicus'' CjMan26A initially be mutagenesis, sequence conservation and kinetics mutant against activated substrate <cite>#4</cite>.
;First 3-D structure: ''Cellvibrio japonicus'' CjMan26A <cite>#6</cite>.

== References ==
<biblio>
#Cartmell2008 pmid=18799462
#2 pmid=15987675
#3 pmid=10742274
#4 pmid=8973192
#5 pmid=12203498
#6 pmid=11382747
#7 pmid=16823793
#8 pmid=18408714
#9 pmid=16171384
#10 pmid=19441796
#Gilkes1991 pmid=1886523
</biblio>

[[Category:Glycoside Hydrolase Families|GH026]]