2011-03-10T11:38:06Z

Mirko Maksimainen:

{{UnderConstruction}}
* [[Author]]s: ^^^Anna Kulminskaya^^^, ^^^Mirko Maksimainen^^^, ^^^Juha Rouvinen^^^
* [[Responsible Curator]]: ^^^Anna Kulminskaya^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH35'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH35.html
|}
</div>


== Substrate specificities ==
The majority of GH35 members are β-galactosidases (EC [{{EClink}}3.2.1.23 3.2.1.23]). GH35 enzymes have been isolated from microorganisms such as fungi, bacteria and yeasts, as well as higher organisms such as plants, animals, and human cells. These β-galactosidases catalyse the hydrolysis of terminal non-reducing β-D-galactose residues in, for example, lactose (1,4-O-β-D-galactopyranosyl-D-glucose), oligosaccharides, glycolipids, and glycoproteins. Various GH35 β-galactosidases demonstrate specificity towards β1,3-, β1,6- or β1,4-galactosidic linkages <cite>Zinin2002, Gamauf2007, Tanthanuch2008</cite>, and are often most active under acidic conditions <cite>Zhang1994, vanCasteren2000, Wang2009</cite>. As with many other CAZy families <cite>GeislerLee2006, Henrissat2001, Tuskan2006</cite>, GH35 members tend to be represented by multi-gene families in plants <cite>Ahn2007, Smith2000, Lazan2004, Ross1994, Tanthanuch2008</cite>. Moreover, plant GH35 β-galactosidases have be divided into two classes: members of the first are capable of hydrolyzing pectic β-1,4-galactans, while those of the second can specifically cleave β-1,3- and β1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>.

In addition to β-galactosidases, GH35 also contains a limited number of archeal exo-β-glucosaminidases (EC [{{EClink}}3.2.1.165 3.2.1.165]) <cite>Tanaka2003 Liu2006</cite>. Such enzymes hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from non-reducing termini.

== Kinetics and Mechanism ==
Beta-galactosidases of GH35 catalyze the hydrolysis of terminal β-galactosyl residues via a double-displacement mechanism, which leads to net retention of the β-anomeric configuration of the released galactose molecule. The stereochemistry of the reaction was first shown by NMR for the human β-galactosidase precursor <cite>Zhang1994</cite> and has been subsequently confirmed by other investigators for microbial and plant enzymes <cite>vanCasteren2000, Zinin2002</cite> .

== Catalytic Residues ==
The catalytic residues for family 35 were first predicted on the basis of hydrophobic cluster analysis of proteins of similar protein fold <cite>Henrissat1995</cite>. Experimentally, the glutamic acid residue 268 was first identified as the catalytic nucleophile in human lysosomal β-galactosidase precursor using a slow substrate, 2,4-dinitrophenyl-2-deoxy-2-fluoro- β-D-galactopyranoside, that allowed trapping of a covalent glycosyl-enzyme intermediate. This allowed subsequent peptide mapping to exactly identify the catalytic nucleophile <cite>McCarter1997</cite>. Subsequently, this approach was repeated for two bacterial β-galactosidases from ''Xanthomonas manihotis'' and ''Bacillus circulans'' <cite>Blanchard2001</cite>. The general acid/base catalyst was inferred to be Glu200 from structural studies of a ''Penicillium'' sp. β-galactosidase <cite>Rojas2004</cite>. Recent structural studies <cite>Maksimainen2010</cite> revealed two different conformations of the general acid/base catalyst in the β-galactosidase of ''Trichoderma reesei''.

== Three-dimensional structures ==
To date (March 2011), only three enzymes from GH35 have been structurally characterized. The first 3D-structures of a GH35 enzyme, those of a β-galactosidase from ''Pencillium'' sp. (Psp-β-gal) in native (PDB [{{PDBlink}}1tg7 1tg7]) and product-complexed (PDB [{{PDBlink}}1xc6 1xc6]) forms, were reported in 2004 at 1.90 Å and 2.10 Å resolution, respectively <cite>Rojas2004</cite>. The structure of a β-galactosidase from ''Bacteriodes thetaiotamicron'' (Btm-β-gal) was subsequently reported by the New York Structural GenomiX Research Consortium in 2008 at 2.15 Å resolution (PDB [{{PDBlink}}3d3a 3d3a]). In 2010, an atomic (1.2 Å) resolution crystal structure of a ''Trichoderma reesei'' (''Hypocrea jecorina'') β-galactosidase (Tr-β-gal, PDB [{{PDBlink}}3og2 3og2]) was reported, together with complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4 Å resolutions, respectively (PDB codes [{{PDBlink}}3ogr 3ogr], [{{PDBlink}}3ogs 3ogs], and [{{PDBlink}}3ogv 3ogv], respectively) <cite>Maksimainen2010</cite>.

The comparison of the native structures of Psp-β-gal, Tr-β-gal and Btmβ-gal shows two things (Figure 1A): Firstly, Btm-β-gal consists of three distinct domains whereas Psp-β-gal and Tr-β-gal consist of five and six domains, respectively. The second and third domains of Btm-β-gal are quite similar with the fourth and fifth domains of Psp-β-gal and with the fifth and sixth domains of Tr-β-gal. Secondly, major structural differences between Psp-β-gal and Tr-β-gal are in the conformations of the loop regions. Although the crystal structures of Psp-β-gal and Tr-β-gal are similar, the interpretation of the structure of Tr-β-gal is somewhat different from that presented earlier for Psp-β-gal: Rojas et al. considered Psp-β-gal to be composed of five distinct structural domains. The overall structure is built around the first, TIM barrel, domain. Domain 2 is an all β-sheet domain containing an immunoglobulin-like subdomain, domain 3 is based on a Greek-key β-sandwich and domains 4 and 5 are jelly rolls <cite>Rojas2004</cite>. In contrast, Maksimainen et al. concluded the domain 2 includes two different domains and thus the Tr-β-gal and Psp-β-gal structures both form six similar domains <cite>Maksimainen2010</cite>.

GH35 enzymes belong to Clan GH-A, and thus have an (α/β)<sub>8</sub> (TIM) barrel as the catalytic domain, in which two glutamic acid residues act as the general acid-base and nucleophilic catalysts. These residues are located in strands 4 and 7 of the barrel. In the crystal structures of Psp-β-gal, Tr-β-gal and Btmβ-gal, the first domain is the catalytic domain. The superimposition of the active sites of the GH35 β-galactosidases shows a remarkable similarity. In addition to the catalytic residues, the active sites of the GH35 β-galactosidases contain many identical residues (Figure 1B). Based on the galactose-bound crystallographic models of Psp-β-gal and Tr-β-gal, a single galactose molecule is bound to the active site of the GH35 enzyme in the chair conformation in the β-anomeric configuration.

Additionally, Maksimainen et al. have described conformational changes in two loop regions of the active site of Tr-β-gal, thus implicating a conformational selection mechanism for the enzyme. Unlike, the induced fit theory which assumes that the initial interaction between a protein and its binding partner induces a conformational change in the protein through a stepwise process, the conformational selection theory is based on the assumption that the unbound protein exists as an ensemble of conformations in dynamic equilibrium. Interaction between a weakly populated, higher-energy conformation and a binding partner causes the equilibrium to move in favor of the selected conformation (Figure 2A) <cite>Tsai1999, Boehr2008</cite>. This can be seen in the structures of Tr-β-gal: the open and closed conformation are both favorable in the native structure and the closed conformation becomes more favorable in the complex structures (Figure 2B,2C). Furthermore, The acid/base catalyst Glu200 has two different conformations in the IPTG and PETG complex structures that clearly affects the p''K''<sub>a</sub> value of this residue and thus the catalytic mechanism of the enzyme <cite>Maksimainen2010</cite>.

=== Structure images ===

<br style="clear: both" />

== Family Firsts ==
;First stereochemistry determination:
Human β-galactosidase precursor by NMR <cite>Zhang1994</cite>

;First catalytic nucleophile identification:
Human β-galactosidase precursor by 2-fluorogalactose labeling <cite>McCarter1997</cite>.

;First general acid/base residue identification:
''Penicillium sp.'' β-galactosidase by structural identification <cite>Rojas2004</cite>.

;First 3-D structure:
''Penicillium sp.'' β-galactosidase <cite>Rojas2004</cite>.

== References ==
<biblio>
#Ahn2007 pmid=17466346
#Smith2000 pmid=10889266
#Lazan2004 pmid=15694277
#Ross1994 pmid=7991682
#Tanthanuch2008 pmid=18664295
#Tanka2003 pmid=12923090
#Liu2006 pmid=16912928
#Zhang1994 pmid=7998946
#Henrissat1995 pmid=7624375
#McCarter1997 pmid=8995274
#Rojas2004 pmid=15491613
#Blanchard2001 pmid=11423106
#Maksimainen2010 Maksimainen M, Hakulinen N, Kallio JM, Timoharju T, Turunen O, Rouvinen J. ''Crystal structures of Trichoderma reesei beta-galactosidase reveal conformational changes in the active site.'' J Struct Biol. 2010, ''in press.'' //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1016/j.jsb.2010.11.024 DOI:10.1016/j.jsb.2010.11.024] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=21130883 PMID:21130883]
#GeislerLee2006 pmid=16415215
#Henrissat2001 pmid=11554480
#Tuskan2006 pmid=16973872
#Gamauf2007 pmid=17381511
#Zinin2002 pmid=11909597
#vanCasteren2000 pmid=11086688
#Wang2009 pmid=19453169
#Kotake2005 pmid=15980190
#Boehr2008 Boehr DD, Wright PE ''How do proteins interact?'' Science 2008, 320 1429-1430.
#Tsai1999 pmid=10468538
</biblio>

[[Category:Glycoside Hydrolase Families|GH035]]

File:GH35 comparison.png

2011-03-10T11:36:57Z

Mirko Maksimainen: uploaded a new version of "File:GH35 comparison.png": Reverted to version as of 11:33, 10 March 2011

File:GH35 comparison.png

2011-03-10T11:36:11Z

Mirko Maksimainen: uploaded a new version of "File:GH35 comparison.png"

2011-03-04T13:34:58Z

Mirko Maksimainen:

{{UnderConstruction}}
* [[Author]]: ^^^Anna Kulminskaya^^^, ^^^Mirko Maksimainen^^^, Juha Rouvinen
* [[Responsible Curator]]: ^^^Anna Kulminskaya^^^
----


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH35'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH35.html
|}
</div>


== Substrate specificities ==
The majority of GH35 members are β-galactosidases (EC [{{EClink}}3.2.1.23 3.2.1.23]). GH35 enzymes have been isolated from microorganisms such as fungi, bacteria and yeasts, as well as higher organisms such as plants, animals, and human cells. These β-galactosidases catalyse the hydrolysis of terminal non-reducing β-D-galactose residues in, for example, lactose (1,4-O-β-D-galactopyranosyl-D-glucose), oligosaccharides, glycolipids, and glycoproteins. Various GH35 β-galactosidases demonstrate specificity towards β1,3-, β1,6- or β1,4-galactosidic linkages <cite>Zinin2002, Gamauf2007, Tanthanuch2008</cite>, and are often most active under acidic conditions <cite>Zhang1994, vanCasteren2000, Wang2009</cite>. As with many other CAZy families <cite>GeislerLee2006, Henrissat2001, Tuskan2006</cite>, GH35 members tend to be represented by multi-gene families in plants <cite>Ahn2007, Smith2000, Lazan2004, Ross1994, Tanthanuch2008</cite>. Moreover, plant GH35 β-galactosidases have be divided into two classes: members of the first are capable of hydrolyzing pectic β-1,4-galactans, while those of the second can specifically cleave β-1,3- and β1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>.

In addition to β-galactosidases, GH35 also contains a limited number of archeal exo-β-glucosaminidases (EC [{{EClink}}3.2.1.165 3.2.1.165]) <cite>Tanaka2003 Liu2006</cite>. Such enzymes hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from non-reducing termini.

== Kinetics and Mechanism ==
Beta-galactosidases of GH35 catalyze the hydrolysis of terminal β-galactosyl residues via a double-displacement mechanism, which leads to net retention of the β-anomeric configuration of the released galactose molecule. The stereochemistry of the reaction was first shown by NMR for the human β-galactosidase precursor <cite>Zhang1994</cite> and has been subsequently confirmed by other investigators for microbial and plant enzymes <cite>vanCasteren2000, Zinin2002</cite> .

== Catalytic Residues ==
The catalytic residues for family 35 were first predicted on the basis of hydrophobic cluster analysis of proteins of similar protein fold <cite>Henrissat1995</cite>. Experimentally, the glutamic acid residue 268 was first identified as the catalytic nucleophile in human lysosomal β-galactosidase precursor using a slow substrate, 2,4-dinitrophenyl-2-deoxy-2-fluoro- β-D-galactopyranoside, that allowed trapping of a covalent glycosyl-enzyme intermediate. This allowed subsequent peptide mapping to exactly identify the catalytic nucleophile <cite>McCarter1997</cite>. Subsequently, this approach was repeated for two bacterial β-galactosidases from ''Xanthomonas manihotis'' and ''Bacillus circulans'' <cite>Blanchard2001</cite>. The general acid/base catalyst was inferred to be Glu200 from structural studies of a ''Penicillium'' sp. β-galactosidase <cite>Rojas2004</cite>. Recent structural studies <cite>Maksimainen2010</cite> revealed two different conformations of the general acid/base catalyst in the β-galactosidase of ''Trichoderma reesei''.

== Three-dimensional structures ==
As of February 2011, only three enzymes from GH35 have been structurally characterized. The first 3D-structures of a GH35 enzyme, those of a β-galactosidase from ''Pencillium'' sp. (Psp-β-gal) in native (PDB [{{PDBlink}}1tg7 1tg7]) and product-complexed (PDB [{{PDBlink}}1xc6 1xc6]) forms, were reported in 2004 at 1.90 Å and 2.10 Å resolution, respectively <cite>Rojas2004</cite>. The structure of a β-galactosidase from ''Bacteriodes thetaiotamicron'' was subsequently reported by the New York Structural GenomiX Research Consortium in 2008 at 2.15 Å resolution (PDB [{{PDBlink}}3d3a 3d3a]). In 2010, a high (1.2 Å) resolution crystal structure of a ''Trichoderma reesei'' (''Hypocrea jecorina'') β-galactosidase (Tr-β-gal, PDB [{{PDBlink}}3og2 3og2]) was reported, together with complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4 Å resolutions, respectively (PDB codes [{{PDBlink}}3ogr 3ogr], [{{PDBlink}}3ogs 3ogs], and [{{PDBlink}}3ogv 3ogv], respectively) <cite>Maksimainen2010</cite>.

GH35 enzymes belong to Clan GH-A, and thus have a (α/β)<sub>8</sub> TIM barrel comprising the catalytic domain. A structural analysis of the galactose-binding active-site was based on the comparison of the crystallographic models of the native Psp-β-gal and Tr-β-gal enzymes and their respective complexes with galactose. A single galactose molecule is bound to the TIM barrel domain of the enzyme in the chair conformation in the β-anomeric configuration. Two glutamic acid residues act as the general acid-base and nucleophilic catalysts; these are presented on strands 4 and 7 of the barrel.Although the crystal structures of Psp-β-gal and Tr-β-gal are similar, interpretation of the structure of Tr-β-gal is somewhat different from that presented earlier for Psp-β-gal: Rojas et al. considered Psp-β-gal to be composed of five distinct structural domains. The overall structure is built around the first, TIM barrel, domain. Domain 2 us an all β-sheet domain containing an immunoglobulin-like subdomain, domain 3 is based on a Greek-key β-sandwich and domains 4 and 5 are jelly rolls <cite>Rojas2004</cite>. In contrast, Maksimainen et al. concluded that the Tr-β-gal and Psp-β-gal structures both form six similar domains and <cite>Maksimainen2010</cite>. The most of the structural differences between them are in the conformations of the loop regions.

Additionally, Maksimainen et al. have described conformational changes in two loop regions of the active site of Tr-β-gal, thus implicating a conformational selection mechanism for the enzyme (Figure 2). The acid/base catalyst Glu200 exhibited two different conformations which affect the p''K''<sub>a</sub> value of this residue and thus the catalytic mechanism.

=== Structure images ===
[[File:GH35 comparison.png|thumb|left|1200px|'''Figure 1. A)''' Comparison of the native structures of the GH35 β-galactosidases. Psp-β-gal (PDB code [http://www.rcsb.org/pdb/explore/explore.do?structureId=1TG7 1tg7]), Tr-β-gal (PDB code [http://www.rcsb.org/pdb/search/structidSearch.do?structureId=3OG2 3og2]) and Btm-β-gal (PDB code [http://www.rcsb.org/pdb/explore/explore.do?structureId=3D3A 3d3a]) are colored in green, brown and blue, respectively. '''B)''' Comparison of the active sites of the GH35 β-galactosidases. Psp-β-gal, Tr-β-gal and Btm-β-gal are colored as above-mentioned.]]

[[File:Conf selection.png|thumb|left|1200px|'''Figure 2. A)''' Molecular recognition mechanisms in proteins: Induced fit vs. conformational selection. The picture is taken from reference <cite>Boehr2008</cite> '''B)''' The native structure of Tr-β-gal. The two conformations (green and blue) of the loop region correspond to the colors of the energy states in Figure 2A. '''C)''' The galactose-bound structure of Tr-β-gal. The galactose molecule (yellow) corresponds to the substrate (triangle) and the purple conformation corresponds to the color of the substrate-bound energy state in Figure 2A.]]

<br style="clear: both" />

== Family Firsts ==
;First stereochemistry determination:
Human β-galactosidase precursor by NMR <cite>Zhang1994</cite>

;First catalytic nucleophile identification:
Human β-galactosidase precursor by 2-fluorogalactose labeling <cite>McCarter1997</cite>.

;First general acid/base residue identification:
''Penicillium sp.'' β-galactosidase by structural identification <cite>Rojas2004</cite>.

;First 3-D structure:
''Penicillium sp.'' β-galactosidase <cite>Rojas2004</cite>.

== References ==
<biblio>
#Ahn2007 pmid=17466346
#Smith2000 pmid=10889266
#Lazan2004 pmid=15694277
#Ross1994 pmid=7991682
#Tanthanuch2008 pmid=18664295
#Tanka2003 pmid=12923090
#Liu2006 pmid=16912928
#Zhang1994 pmid=7998946
#Henrissat1995 pmid=7624375
#McCarter1997 pmid=8995274
#Rojas2004 pmid=15491613
#Blanchard2001 pmid=11423106
#Maksimainen2010 Maksimainen M, Hakulinen N, Kallio JM, Timoharju T, Turunen O, Rouvinen J. ''Crystal structures of Trichoderma reesei beta-galactosidase reveal conformational changes in the active site.'' J Struct Biol. 2010, ''in press.'' //''Note: Due to a problem with PubMed data, this reference is not automatically formatted. Please see these links out:'' [http://dx.doi.org/10.1016/j.jsb.2010.11.024 DOI:10.1016/j.jsb.2010.11.024] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=21130883 PMID:21130883]
#GeislerLee2006 pmid=16415215
#Henrissat2001 pmid=11554480
#Tuskan2006 pmid=16973872
#Gamauf2007 pmid=17381511
#Zinin2002 pmid=11909597
#vanCasteren2000 pmid=11086688
#Wang2009 pmid=19453169
#Kotake2005 pmid=15980190
#Boehr2008 Boehr DD, Wright PE ''How do proteins interact?'' Science 2008, 320 1429-1430.
</biblio>

[[Category:Glycoside Hydrolase Families|GH035]]