Glycoside Hydrolase Family 35 - Revision history

Harry Brumer: Added PMID for Boehr reference

2026-03-20T16:25:30Z

Added PMID for Boehr reference

Harry Brumer at 16:22, 20 March 2026

2026-03-20T16:22:14Z

Masahiro Nakajima at 15:54, 20 March 2026

2026-03-20T15:54:14Z

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:18:23Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Karen Eddy at 22:11, 14 June 2012

2012-06-14T22:11:26Z

Spencer Williams at 01:51, 25 March 2011

2011-03-25T01:51:10Z

Anna Kulminskaya at 09:22, 21 March 2011

2011-03-21T09:22:25Z

Harry Brumer at 09:05, 21 March 2011

2011-03-21T09:05:23Z

Harry Brumer: updated Maksimainen ref.

2011-03-21T09:04:08Z

updated Maksimainen ref.

Harry Brumer at 08:58, 21 March 2011

2011-03-21T08:58:19Z

@@ Line 94: / Line 94: @@
 #Wang2009 pmid=19453169
 #Kotake2005 pmid=15980190
-#Boehr2008 Boehr DD, Wright PE ''How do proteins interact?'' Science 2008, 320 1429-1430.
+#Boehr2008 pmid=18556537
 #Tsai1999  pmid=10468538
 #Nakazawa2025 pmid=39820076

@@ Line 29: / Line 29: @@
 == Substrate specificities ==
-The majority of [[glycoside hydrolases]] of GH35 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 β-1,3-, β-1,6- or  β-1,4-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 β-1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>. In 2025, β-galactosidase acting on β-1,2-galactooligosaccharides specifically (EC [{{EClink}}3.2.1.230 3.2.1.230]) was reported <cite>Nakazawa2025</cite>.
+The majority of [[glycoside hydrolases]] of GH35 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 β-1,3-, β-1,6- or  β-1,4-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 β-1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>. In 2025, a β-galactosidase acting specifically on β-1,2-galactooligosaccharides (EC [{{EClink}}3.2.1.230 3.2.1.230]) was reported <cite>Nakazawa2025</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> and β-1,2-glucosyltransferase (EC [{{EClink}}2.4.1.391 2.4.1.391]) <cite>Kobayashi2022</cite>. The former enzymes hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from non-reducing termini. The latter enzyme disproportionates β-1,2-glucooligosaccharides by transferring glucose units but prefers sophorose (Glc-β-1,2-Glc) as a donor and alkyl- or acyl-glucosides as acceptors, regardless of their anomeric configuration.

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]s: [[User:Anna Kulminskaya|Anna Kulminskaya]], [[User:Mirko Maksimainen|Mirko Maksimainen]], [[User:Juha Rouvinen|Juha Rouvinen]]
+* [[Author]]s: [[User:Anna Kulminskaya|Anna Kulminskaya]], [[User:Mirko Maksimainen|Mirko Maksimainen]], [[User:Juha Rouvinen|Juha Rouvinen]], [[User:Masahiro Nakajima|Masahiro Nakajima]]
 * [[Responsible Curator]]:  [[User:Anna Kulminskaya|Anna Kulminskaya]]
 ----
@@ Line 29: / Line 29: @@
 == Substrate specificities ==
-The majority of [[glycoside hydrolases]] of GH35 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 β-1,3-, β-1,6- or  β-1,4-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 β-1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>.
+The majority of [[glycoside hydrolases]] of GH35 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 β-1,3-, β-1,6- or  β-1,4-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 β-1,6-galactosyl linkages of arabinogalactan proteins <cite>Kotake2005</cite>. In 2025, β-galactosidase acting on β-1,2-galactooligosaccharides specifically (EC [{{EClink}}3.2.1.230 3.2.1.230]) was reported <cite>Nakazawa2025</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.
+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> and β-1,2-glucosyltransferase (EC [{{EClink}}2.4.1.391 2.4.1.391]) <cite>Kobayashi2022</cite>. The former enzymes hydrolyze chitosan or chitosan oligosaccharides to remove successive D-glucosamine residues from non-reducing termini. The latter enzyme disproportionates β-1,2-glucooligosaccharides by transferring glucose units but prefers sophorose (Glc-β-1,2-Glc) as a donor and alkyl- or acyl-glucosides as acceptors, regardless of their anomeric configuration.
 == Kinetics and Mechanism ==
@@ Line 96: / Line 96: @@
 #Boehr2008 Boehr DD, Wright PE ''How do proteins interact?'' Science 2008, 320 1429-1430.
 #Tsai1999  pmid=10468538
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH035]]

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]s: ^^^Anna Kulminskaya^^^, ^^^Mirko Maksimainen^^^, ^^^Juha Rouvinen^^^
+* [[Author]]s: [[User:Anna Kulminskaya|Anna Kulminskaya]], [[User:Mirko Maksimainen|Mirko Maksimainen]], [[User:Juha Rouvinen|Juha Rouvinen]]
-* [[Responsible Curator]]:  ^^^Anna Kulminskaya^^^
+* [[Responsible Curator]]:  [[User:Anna Kulminskaya|Anna Kulminskaya]]
 ----

@@ Line 85: / Line 85: @@
 #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. Apr; 174(1): 156-63. //''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]
+#Maksimainen2010 pmid=21130883
 #GeislerLee2006 pmid=16415215
 #Henrissat2001 pmid=11554480

@@ Line 29: / Line 29: @@
 == 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>.
+The majority of [[glycoside hydrolases]] of GH35 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 β-1,3-, β-1,6- or  β-1,4-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 β-1,6-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.
+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> .
+Beta-galactosidases of GH35 catalyze the hydrolysis of terminal β-galactosyl residues via a [[classical Koshland retaining 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 and subsequent peptide mapping <cite>McCarter1997</cite>. 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 (''vide infra'') revealed two different conformations of the general acid/base catalyst in the β-galactosidase of ''Trichoderma reesei'' <cite> Maksimainen2010</cite>.
+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, which allowed trapping of a covalent glycosyl-enzyme intermediate and subsequent peptide mapping <cite>McCarter1997</cite>. This approach was repeated for two bacterial β-galactosidases from ''Xanthomonas manihotis'' and ''Bacillus circulans'' <cite>Blanchard2001</cite>. The [[general acid/base]] residue was inferred to be Glu200 from structural studies of a ''Penicillium'' sp. β-galactosidase <cite>Rojas2004</cite>. Recent structural studies (''vide infra'') revealed two different conformations of the [[general acid/base]] residue in the β-galactosidase of ''Trichoderma reesei'' <cite> Maksimainen2010</cite>.
 == Three-dimensional structures ==
@@ Line 60: / Line 60: @@
 == Family Firsts ==
 ;First stereochemistry determination:
-Human β-galactosidase precursor by NMR <cite>Zhang1994</cite>
+[[Retaining]] stereochemical outcome for human β-galactosidase precursor by NMR <cite>Zhang1994</cite>
-;First catalytic nucleophile identification:
+;First [[catalytic nucleophile]] identification:
 Human β-galactosidase precursor by 2-fluorogalactose labeling <cite>McCarter1997</cite>.
-;First general acid/base residue identification:
+;First [[general acid/base]] residue identification:
 ''Penicillium sp.'' β-galactosidase by structural identification <cite>Rojas2004</cite>.

@@ Line 37: / Line 37: @@
 == 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''.
+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 and subsequent peptide mapping <cite>McCarter1997</cite>. 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 (''vide infra'') revealed two different conformations of the general acid/base catalyst in the β-galactosidase of ''Trichoderma reesei''<cite> Maksimainen2010</cite>.
 == Three-dimensional structures ==
 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.
-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.
+The comparison of the native structures of Psp-β-gal, Tr-β-gal and Btmβ-gal reveals two things ('''Figure 1'''): 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>.
 Additionally, Maksimainen et al. have described conformational changes in two loop regions of the active site of Tr-β-gal, that implicates a conformational selection mechanism for the enzyme (Figure 2). 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 <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. 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>.