CAZypedia - User contributions [en-ca]

User:Kazune Tamura

2022-01-12T03:59:32Z

Kazune Tamura:

[[Image:IMG 2698.jpeg|200px|right]]
Kazune Tamura obtained his B.Sc. in Combined Honours in Biochemistry and Chemistry from the University of British Columbia in 2015. During this time, he completed an undergraduate thesis project in the lab of Dr. [[User:Harry Brumer|Harry Brumer]] where he stayed to complete his Ph.D. focused around the study of microbial utilization of common beta-glucans via polysaccharide utilization loci <cite>Tamura2017 Grondin2017 Tamura2019 Dejean2020 Tamura2021a Tamura2021b Jain2021 Behar2021 Golisch2021</cite>. He now applies his expertise in carbohydrate biochemistry and structural biology to the investigation of mechanisms of cell-cell adhesion in bacterial biofilms at the University of Oxford and MRC-LMB.

----

<biblio>
#Tamura2017 pmid=29020628
#Grondin2017 pmid=28138099
#Tamura2019 pmid=31062073
#Dejean2020 pmid=32265336
#Tamura2021a pmid=33587952
#Tamura2021b pmid=33285501
#Jain2021 pmid=33988963
#Behar2021 pmid=34338284
#Golisch2021 pmid=34709792

</biblio>


[[Category:Contributors|Tamura,Kazune]]

Glycoside Hydrolase Family 28

2020-05-22T22:55:19Z

Kazune Tamura:

User:Kazune Tamura

2020-04-11T01:07:37Z

Kazune Tamura:

Kazune Tamura: /* Three-dimensional structures */

{{CuratorApproved}}
* [[Author]]: [[User:Withers|Stephen Withers]]
* [[Responsible Curator]]: [[User:Withers|Stephen Withers]]
----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH2'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH2.html
|}
</div>

== Substrate specificities ==
The most common activities for [[glycoside hydrolases]] of this family include β-galactosidases, β-glucuronidases, β-mannosidases, [[exo]]-β-glucosaminidases and, in plants, a mannosylglycoprotein [[endo]]-β-mannosidase. The enzymes are found across a broad spectrum of life forms, but are concentrated in bacteria. The most famous enzyme in this family is the ''E. coli'' (lacZ) β-galactosidase, a component of the lac operon. Not only did this enzyme play a key role in developing the understanding of operon structure and control of gene expression, but also it continues to play a key role as a cell biological probe. Another matter of note is that this remains the largest protein monomer to be sequenced entirely at the peptide level <cite>FowlerZabin1978</cite>. ''E. coli'' also contains a second, vestigial β-galactosidase (ebg) whose activity has been shown to evolve in lacZ<sup>-</sup> strains of ''E. coli'' grown under selective pressure with lactose as sole carbon source <cite>Hall1999 Krishnan1995</cite>. Another reasonably well-studied GH2 enzyme is the ''E. coli'' β-glucuronidase, whose activity is used to detect the presence of ''E. coli'' (http://www.cfsan.fda.gov/~ebam/bam-4.html), though interestingly not the nasty O157 strain. The principal enzyme of medical interest in GH2 is the lysosomal β-glucuronidase whose deficiency leads to Sly syndrome <cite>Sly1973</cite>. The only other human GH2 enzyme is the lysosomal β-mannosidase.

== Kinetics and Mechanism ==
Family 2 β-glycosidases are [[retaining]] enzymes and follow a classical [[Koshland double-displacement mechanism]]. This was first evidenced in 1951 by Wallenfels, who demonstrated the transglycosylation of lactose via an implicated glycosyl-enzyme intermediate <cite>Wallenfels1951</cite> The best studied enzyme kinetically must be the ''E. coli'' (lacZ) β-galactosidase, for which a key set of studies defining the two-step mechanism and elucidating rate-limiting steps was published by the groups of Yon and Sinnott in the early 1970’s <cite>SinnottSouchard1973 SinnottViratelle1973 ViratelleYon1973</cite>. Indeed the approaches developed on that system laid the foundations for many subsequent studies on other glycosidases. An analysis of the roles of each substrate hydroxyl in catalysis, based upon kinetic studies with modified sugars has also been published <cite>McCarter1992</cite>. Some GH2 glycosidases require Mg<sup>2+</sup> for activity and in ''E. coli'' β-galactosidase this Mg<sup>2+</sup> requirement is associated with the binding of the cation in the active site such that it places the acid/base residue appropriately. Others, such as the human β-glucuronidase, have no such metal ion requirement.

== Catalytic Residues ==
The [[catalytic nucleophile]] in GH2 was first correctly identified in the ''E. coli'' (lacZ) β-galactosidase as Glu537 in the sequence ILC'''<u>E</u>'''YAH through trapping of the 2-deoxy-2-fluorogalactosyl-enzyme [[intermediate]] and subsequent peptide mapping via HPLC techniques using radiolabeled tracers <cite>Gebler1992</cite>. Earlier studies, carefully done using conduritol C cis-epoxide as affinity label, had identified Glu461 as the labeled residue, <cite>HerrchenLegler1984</cite> on which basis a series of beautifully executed kinetic studies were performed on mutants modified at this position that appeared initially to support this conclusion <cite>Cupples1990</cite>. However, doubts were raised when similar kinetic analysis of nucleophile mutants of the GH1 Agrobacterium sp. β-glucosidase yielded quite different results, leading to the above labeling study <cite>Gebler1992</cite>. The [[general acid/base]] catalyst was then identified as Glu461 by re-interpretation <cite>Gebler1992</cite> of the published kinetic results on mutants at that position <cite>Cupples1990</cite>, which had included azide rescue experiments. These conclusions were fully supported by subsequent 3-dimensional structural analyses (below).

== Three-dimensional structures ==
Three-dimensional structures are available for five Family GH2 enzymes currently, the first solved being that of the ''E. coli'' (lacZ) β-galactosidase in a ''tour de force'' of X-ray crystallography at that time, given its huge size (4 x 125,000 Da) <cite>Jacobson1994</cite>. The enzyme is multidomain, but as members of Clan GHA the catalytic domain is a classical (α/β)<sub>8</sub> 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).

== Family Firsts ==
;First stereochemistry determination: Via transglycosylation of lactose <cite>Wallenfels1951</cite>
;First [[catalytic nucleophile]] identification: ''E. coli'' (lacZ) β-galactosidase by 2-fluorogalactose labeling <cite>Gebler1992</cite>
;First [[general acid/base]] residue identification: ''E. coli'' (lacZ) β-galactosidase by re-interpretation of kinetic studies with mutants <cite>Gebler1992 Cupples1990</cite>
;First 3-D structure: ''E. coli'' (lacZ) β-galactosidase <cite>Jacobson1994</cite>

== References ==
<biblio>
#FowlerZabin1978 pmid=97298
#Hall1999 pmid=10234816
#Sly1973 pmid=4265197
#Wallenfels1951 Wallenfels K. ''Enzymatische synthese von oligosacchariden aus disacchariden.'' Naturwissenschaften, 1951; 38: 306-307. [http://dx.doi.org/10.1007/BF00636782 DOI:10.1007/BF00636782]
#SinnottSouchard1973 pmid=4578762
#SinnottViratelle1973 pmid=4721624
#ViratelleYon1973 pmid=4691347
#McCarter1992 pmid=1417731
#Gebler1992 pmid=1350782
#HerrchenLegler1984 pmid=6420154
#Cupples1990 pmid=1969405
#Jacobson1994 pmid=8008071
#Krishnan1995 pmid=8554546
</biblio>

[[Category:Glycoside Hydrolase Families|GH002]]

Glycoside Hydrolase Family 147

2019-02-23T23:34:41Z

Kazune Tamura: /* Kinetics and Mechanism */

Glycoside Hydrolase Family 50

2019-02-23T23:28:56Z

Kazune Tamura: /* Kinetics and Mechanism */

Glycoside Hydrolase Family 39

2019-02-23T23:27:42Z

Kazune Tamura: /* Substrate Specificities */

{{CuratorApproved}}

* [[Author]]s: ^^^Brian Rempel^^^ and ^^^Darryl Jones^^^
* [[Responsible Curator]]: ^^^Steve Withers^^^

----

<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family 39'''
|-
|'''Clan'''
|GH-A
|-
|'''Mechanism'''
|retaining
|-
|'''Active site residues'''
|known
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH39.html
|}
</div>

==Substrate Specificities==
This [[glycoside hydrolase]] family predominantly consists of two known enzyme activities: β-xylosidase and α-L-iduronidase. Both enzyme activities cleave equatorial glycosidic bonds: the 'α' designation of α-iduronidase is a consequence of the stereochemical designations used for carbohydrates in which the α/β designation is related to the [[Absolute_configuration:_D/L_nomenclature|D/L designation]] defined by the stereochemistry at C5 in hexopyranoses <cite>McNaught1997</cite>. In addition to β-xylosidase activity, the equatorial bond cleaving β-glucosidase, β-galactosidase, and xylanase activities have been identified in one family GH39 enzyme <cite>Morrison2016</cite>. Furthermore, recent studies have characterized a GH39 from ''Pseudomonas aeruginosa'' active on the exopolysaccharide Psl (composed of D-mannose, D-glucose, and L-rhamnose) <cite>Baker2015 Byrd2009</cite>, and a group of fungal GH39 enzymes which possess α-L-(β-1,2)-arabinobiosidase activity and can release D-galactose-(α-1,2)-L-arabinose from arabinoxylans <cite>Jones2017</cite>. Enzymes from this family are currently found in bacteria and eukaryotes, although eleven gene sequences encoding putative Family GH39 enzymes from archaea have been reported in the CAZy database. The known β-xylosidase enzymes for which an enzyme activity has been experimentally established all come from microbes, while the α-iduronidase enzymes all come from metazoan eukaryotes. Additionally, while there is a reasonable degree of sequence similarity within the bacterial β-xylosidases in GH39 and within the α-iduronidases in GH39 <cite>Vocadlo1998</cite>, there is a much lower degree of homology between enzymes with differing activities <cite>Vocadlo1998 Baker2015 Jones2017 Ali-Ahmad2017</cite>. The best-studied enzymes are human α-iduronidase, whose deficiency causes Mucopolysaccharidosis I (also known as Hurler-Scheie syndrome), and the β-xylosidase from ''Thermoanaerobacterium saccharolyticum''.

==Kinetics and Mechanism==
Family GH39 enzymes are [[retaining]] [[glycoside hydrolases]] that follow the classical [[Koshland double-displacement mechanism]]. This has been demonstrated experimentally through NMR analysis of the first-formed sugar product produced by glycoside hydrolysis by the β-xylosidase from ''Thermoanaerobacterium saccharolyticum'' <cite>Armand1996</cite> and human α-iduronidase <cite>Nieman2003</cite>, and by covalent trapping of the [[catalytic nucleophile]] (described below) for these two enzymes <cite>Vocadlo1998 Nieman2003</cite>. These enzymes do not appear to require any activator or cofactor for activity.

==Catalytic Residues==
The [[catalytic nucleophile]] was first identified in the β-xylosidase from ''Thermoanaerobacterium saccharolyticum'' as Glu-277 in the sequence IILNSHFPNLPFHIT<u>'''E'''</u>Y by trapping of the 2-deoxy-2-fluoro-xylosyl-enzyme [[intermediate]] and subsequent peptide mapping by LC/MS-MS <cite>Vocadlo1998</cite>. A similar analysis performed on human α-iduronidase also successfully trapped the [[catalytic nucleophile]] and identified it as Glu-299 in the sequence IYND<u>'''E'''</u>AD <cite>Nieman2003</cite>, which confirmed previous theoretical predictions <cite>Durnad1997</cite>. The [[general acid/base]] residue has been experimentally identified in the β-xylosidase from ''Thermoanaerobacterium saccharolyticum'' as Glu-160 through trapping using the affinity label N-bromoacetyl-β-D-xylopyranosylamine and analysis of variant proteins created by mutation of that site <cite>Vocadlo2002</cite>.

==Three-dimensional structures==
The three-dimensional structure of the β-xylosidase from ''Thermoanaerobacterium saccharolyticum'' was first solved in 2004 <cite>Yang2004</cite>. Since then, the three dimensional structures for GH39 enzymes from ''Geobacillus stearothermophilus'' <cite>Czjzek2005 Czjzek2004</cite>, ''Homo sapiens'' <cite>Maita2013 Bie2013</cite>, ''Pseudomonas aeruginosa'' <cite>Baker2015</cite>, ''Neocallimastix frontalis'' <cite>Jones2017</cite>, and ''Bacteroides cellulosilyticus'' <cite>Ali-Ahmad2017</cite> have also been solved. GH39 enzymes are members of the [[Sequence-based classification of glycoside hydrolases|clan]] GH-A fold, consistent with the classic (α/β)<sub>8</sub> 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).

==Family Firsts==

;'''First stereochemistry determination'''
:''Thermoanaerobacterium saccharolyticum'' β-xylosidase by NMR <cite>Armand1996</cite>

;'''First [[catalytic nucleophile]] identification'''
:''Thermoanaerobacterium saccharolyticum'' β-xylosidase by 2-fluoroxylose labelling <cite>Nieman2003</cite>

;'''First [[general acid/base]] residue identification'''
:''Thermoanaerobacterium saccharolyticum'' β-xylosidase through labelling with N-bromoacetyl-β-D-xylopyranosylamine and kinetic analysis of mutants generated at the identified position <cite>Durnad1997</cite>

;'''First 3-D structure of a GH39 enzyme'''
:''Thermoanaerobacterium saccharolyticum'' β-xylosidase <cite>Vocadlo2002</cite>

==References==
<biblio>
#McNaught1997 pmid=9042704
#Vocadlo1998 pmid=9761746
#Armand1996 pmid=8612648
#Nieman2003 pmid=12834357
#Durnad1997 pmid=9134434
#Vocadlo2002 pmid=12146939
#Yang2004 pmid=14659747
#Czjzek2005 pmid=16212978
#Czjzek2004 pmid=14993701
#Morrison2016 pmid=27547582
#Baker2015 pmid=26424791
#Byrd2009 pmid=19659934
#Jones2017 pmid=28588026
#Ali-Ahmad2017 pmid=27890857
#Maita2013 pmid=23959878
##Bie2013 pmid=24036510

</biblio>

[[Category:Glycoside Hydrolase Families|GH039]]

Glycoside Hydrolase Family 6

2019-02-23T23:26:22Z

Kazune Tamura: /* Three-dimensional structures */

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


<div style="float:right">
{| {{Prettytable}}
|-
|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH6'''
|-
|'''Clan'''
|none
|-
|'''Mechanism'''
|inverting
|-
|'''Active site residues'''
|acid known, base debated
|-
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
|-
| colspan="2" |{{CAZyDBlink}}GH6.html
|}
</div>


== Substrate specificities ==
[[Glycoside hydrolases]] of family GH6 cleave β-1,4 glycosidic bonds in cellulose / β-1,4-glucans. Only endoglucanase (EC [{{EClink}}3.2.1.4 3.2.1.4]) and cellobiohydrolase (EC [{{EClink}}3.2.1.91 3.2.1.91]) activity has been reported for the bacterial and eukaryotic members of this family. GH6 was one of the first glycoside hydrolase families classified by hydrophobic cluster analysis, and was previously known as "Cellulase Family B" <cite>Henrissat1989 Gilkes1991</cite>.

== Kinetics and Mechanism ==
GH6 enzymes perform catalysis with [[inverting|inversion]] of anomeric stereochemistry, as first shown by NMR <cite>Knowles1988</cite> on cellobiohydrolase II (CBH II; Cel6A) from the fungus ''Trichoderma reesei'' (a clonal derivative of ''Hypocrea jecorina'' <cite>Kuhls1996</cite>).

== Catalytic Residues ==
The first 3D structure of CBHII provided a strong clue as to the identification of the catalytic [[general acid]] in the [[inverting]] mechanism (Asp 221 in the case of this ''Trichoderma reesei'' Cel6A enzyme). This assigment has withstood the tests of time with strong kinetic support (from kinetics as a function of leaving group ability for a series of enzyme variants) <cite>Damude1995</cite> as well as from all subsequent 3-D analyses of enzyme-ligand complexes (for example <cite>Zou1999 Varrot2005 Varrot2002</cite> ). The identification of the catalytic [[general base]] is, however, far less clear. Simply put this is because there is no clear potential base within hydrogen-bonding distance of a water molecule that could act as the nucleophile in the [[inverting]] mechanism. Thus, although there are mutagenesis / kinetic proposals for a base <cite>Damude1995</cite>, the current 'Zeitgeist' is that the attacking water is deprotonated via a string of water molecules in what Sinnott has descibed as a "Grotthuss" mechanism; for which there is solvent kinetic isotope effect support <cite>Koivula2002</cite>. On the basis of structure the residue most likely to act as the [[general base]] is Asp175 on the ''Trichoderma reesei'' Cel6A, although Asp401 may also play a role (see Table 1).

----
{| {{Prettytable}} style="text-align:left"
|+ Table 1. Putative catalytic residues of some representatives in GH family 6<br>(with biochemical characterization of wt and mutant enzymes).
! Proposed role
! ''Cf''Cel6A (endo)
! ''Hi''Cel6A (exo)
! ''Hj''Cel6A (exo)
! ''Tf''Cel6A (endo)
! ''Tf''Cel6B (exo)
|-
| Substrate distortion
| Tyr210
| Tyr174
| Tyr169
| Tyr73
| Tyr220
|-
| Increase in pKa acid/Catalytic base
| Asp216
| Asp180
| Asp175
| Asp79
| Asp226
|-
| Proton network
| Gly222?
| Ser186
| Ser181
| Ser85
| Ser232
|-
| Catalytic acid
| Asp252
| Asp226
| Asp221
| Asp117
| Asp274
|-
| Catalytic base/substrate binding
| Asp392
| Asp405
| Asp401
| Asp265
| Asp497
|}
----

== Three-dimensional structures ==
The first crystal structures of cellobiohydrolases and endoglucanases from family [[GH6]] revealed modified α/β barrel folds which, unlike the classical (β/α)<sub>8</sub> "TIM" barrel has just seven β-strands forming the central β-barrel. The CBHII structure revealed an active centre (see above) enclosed in a tunnel formed primarily by two surface loops. When, subsequently, the first endoglucanase from GH6 was solved, the active center was observed in a long open groove. The comparison of these two structures thus provided the first insight into how endo or processive activity was modulated, through display of the active centre in a in an open grove, or loop-enclosed tunnel, respectively. In 1995 the UBC group were able to truncate the extended loops of a cellobiohydrolase resulting in an enzyme with more endo-activity <cite>Meinke1995</cite>. To this day the debate continues about the possibilities of loop conformational change in moderating the activity of cellobiohydrolases between exo and endo. Ståhlberg was perhaps the first to explicitly state that ''T. reesei'' "has no true exo-cellulases" <cite>Stberg1993</cite>. It is clear that there is no absolute steric demand for the ''exo'' activity of cellobiohydrolases; the enzymes have a viable "-3" subsite <cite>Varrot1999, Varrot2003</cite>, the loops of the cellobiohydrolases are clearly mobile and show multiple conformations (consistent with occasional opening to support ''endo''-activity, and are also able to act on artificial substrates in which both ends have large appended groups (for example <cite>Armand1997</cite>).

The nature of how catalysis was achieved, and the conformational itinerary of catalysis was first provided by the Uppsala, Grenoble and Gent groups in 1999 <cite>Zou1999</cite> was a trapped Michaelis complex of a thio-oligosaccharide was observed spanning the active centre with the -1 subsite sugar in <sup>2</sup>''S''<sub>O</sub> conformation, which suggested a pathway around the <sup>2,5</sup>''B'' [[transition state]] conformation. Subsequent structural <cite>Varrot2005 Varrot2002</cite> and modelling <cite>Koivula2002</cite> support for these proposals comes from similarly distorted species on other GH6 enzymes and from the observation of a "cellobiosyl isofagomine" in <sup>2,5</sup>''B'' conformation <cite>Varrot2003</cite>.

== Family Firsts ==
;First sterochemistry determination: ''Hypocrea jecorina'' cellobiohydrolase Cel6A by NMR <cite>Knowles1988</cite>.
;First [[general acid]] residue identification: The role of Asp221 as the potential catalytic acid was first proposed on the basis of 3-D structure of the ''Hypocrea jecorina'' cellobiohydrolase CBHII / Cel6A <cite>Rouvinen1990</cite>. Enzyme kinetics of variants, in conjunction with leaving groups requiring provided strong confirmation <cite>Damude1995</cite>.
;First [[general base]] residue identification: The existence / identification of the catalytic base is less clear and current beliefs are that the water is deprotonated through a "solvent wire" through to one of the conserved aspartates near the active centre.
;First 3-D structure: The catalytic core domain of the ''Trichoderma reesei'' (the organism now known as ''Hypocrea jecorina'') cellobiohydrolase II by the Jones group <cite>Rouvinen1990</cite>. The first endoglucanase in this family was the ''Thermomonospora fusca'' E2 enzyme (catalytic core) solved by the Wilson/Karplus groups<cite>Spezio1993</cite>

== References ==
<biblio>
#Knowles1988 Knowles, J.K.C., Lehtovaara, P., Murray, M. and Sinnott, M.L. (1988) Stereochemical course of the action of the cellobioside hydrolases I and II of ''Trichoderma reesei''. J. Chem. Soc., Chem. Commun., 1988, 1401-1402. [http://dx.doi.org/10.1039/C39880001401 DOI: 10.1039/C39880001401]
#Kuhls1996 pmid=8755548
#Rouvinen1990 pmid=2377893
#Spezio1993 pmid=8399160
#Koivula2002 pmid=12188666
#Zou1999 pmid=10508787
#Varrot2003 pmid=12744312
#Varrot2005 pmid=15824123
#Varrot2002 pmid=12454501
#Varrot1999 pmid=10413461
#Damude1995 pmid=7857933
#Meinke1995 pmid=7876202
#Armand1997 pmid=9006908
#Stberg1993 pmid=8499476
#Henrissat1989 pmid=2806912
#Gilkes1991 pmid=1886523
</biblio>

[[Category:Glycoside Hydrolase Families|GH006]]

User:Kazune Tamura

2019-02-23T23:21:22Z

Kazune Tamura:

[[Image:Blank_user-200px.png|200px|right]]
Kazune Tamura obtained his B.Sc. in Combined Honours in Biochemistry and Chemistry from the University of British Columbia in 2015. During this time, he completed an undergraduate thesis project in the lab of Dr. Harry Brumer where he has stayed on as a Ph.D. candidate (class of 2020). His current work is focused around the study of microbial utilization of common beta-glucans via polysaccharide utilization loci<cite>Tamura2017 Grondin2017</cite>.

----

<biblio>
#Tamura2017 pmid=29020628
#Grondin2017 pmid=28138099

</biblio>


[[Category:Contributors|Tamura,Kazune]]