Glycoside Hydrolase Family 99 - Revision history

Spencer Williams: /* Kinetics and Mechanism */ fixed figure legend

2022-09-17T07:27:22Z

Kinetics and Mechanism: fixed figure legend

Spencer Williams: /* Three-dimensional structures */

2022-09-17T07:26:01Z

Three-dimensional structures

Spencer Williams: /* Catalytic Residues */

2022-09-17T07:22:10Z

Catalytic Residues

Spencer Williams: /* Three-dimensional structures */

2022-09-17T07:21:03Z

Three-dimensional structures

Spencer Williams: /* Family Firsts */

2022-09-17T07:20:23Z

Family Firsts

Spencer Williams: /* References */

2022-09-17T07:20:09Z

References

Spencer Williams: /* References */

2022-09-17T07:19:55Z

References

Spencer Williams: /* Family Firsts */

2022-09-17T07:19:29Z

Family Firsts

Spencer Williams: /* Three-dimensional structures */ added detail on MANEA structure

2022-09-17T07:17:57Z

Three-dimensional structures: added detail on MANEA structure

Spencer Williams: /* References */

2022-09-17T06:59:19Z

References

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 Family GH99 [[endo]]-α-mannosidases are [[retaining]] enzymes, as first shown by <sup>1</sup>H NMR analysis of the hydrolysis of &alpha;-glucosyl-1,3-&alpha;-mannopyranosyl fluoride by ''Bacteroides thetaiotaomicron'' <cite>Thompson2012</cite>. [[Retaining]] mannoside hydrolases (eg [[GH38]]) typically follow a [[classical Koshland double-displacement mechanism]]. However, X-ray crystallographic analysis of ''Bx''GH99 and ''Bt''GH99 failed to reveal a candidate nucleophilic residue; instead an alternative mechanism involving substrate-assisted catalysis by the 2OH residue and proceeding through a 1,2-anhydro sugar was proposed <cite>Thompson2012</cite>. In this proposal, Glu333 in ''BxGH99'' (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar. Strong evidence to support this proposed intermediate has been obtained through kinetic isotope effect measurements. In particular, KIE effects on ''k''<sub>cat</sub>/''K''<sub>M</sub> for anomeric-<sup>2</sup>H and anomeric-<sup>13</sup>C support an oxocarbenium ion-like transition state, and that for C2-<sup>18</sup>O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen <cite>Sobala2020</cite>. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar <cite>Sobala2020</cite>.
-[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Proposed catalytic mechanisms for GH99 endo-&alpha;-mannosidase. (A) [[Classical Koshland double-displacement mechanism]]. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.''']]
+[[Image:GH99_mechanisms.png|thumb|center|800px|'''Figure 2. Catalytic mechanism for GH99 endo-&alpha;-mannosidase/endo-&alpha;-mannanase. (A) [[Classical Koshland double-displacement mechanism]] of a typical retaining glycosidase. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate for family GH99.''']]
 == Catalytic Residues ==

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 The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.
-which was observed
 A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.

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 == Catalytic Residues ==
-The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate &alpha;-glucosyl-1,3-&alpha;-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc<sub>3</sub>Man<sub>7</sub>GlcNAc<sub>2</sub>, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the identity of the nucleophilic residue used for catalysis is more obscure and the 2OH of the substrate has been proposed to act as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012</cite>. According to this proposal, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.
+The [[general acid/base]] was highlighted by X-ray crystallographic analysis as Glu336 in ''Bx''GH99 and Glu332 in ''Bt''GH99 <cite>Thompson2012</cite>. The Glu332Ala mutant of ''Bt''GH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate &alpha;-glucosyl-1,3-&alpha;-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc<sub>3</sub>Man<sub>7</sub>GlcNAc<sub>2</sub>, supporting the role of this residue as [[general acid/base]]. As described in "Kinetics and Mechanism" the enzyme lacks a nucleophilic residue used for catalysis as the 2OH of the substrate acts as a nucleophile in a mechanism involving substrate assisted catalysis <cite>Thompson2012 Sobala2020A</cite>. According to this mechanism, Glu333 in ''Bx''GH99 (Glu329 in ''Bt''GH99) acts as a [[general acid/base]] to deprotonate the 2OH and protonate the 1,2-anhydrosugar.
 == Three-dimensional structures ==

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 == Three-dimensional structures ==
-Three-dimensional structures are available for human MANEA <cite>Sobala2020</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (&beta;/&alpha;)<sub>8</sub> TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate.  Binary complexes with two inhibitors, &alpha;-glucosyl-1,3-deoxymannonojirimycin and &alpha;-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-&alpha;-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.
+Three-dimensional structures are available for human MANEA <cite>Sobala2020P</cite>, and bacterial members of GH99, including ''B. thetaiotaomicron'' and ''B. xylanisolvens'' <cite>Thompson2012</cite>. They have a classical (&beta;/&alpha;)<sub>8</sub> TIM barrel fold, which is distinguished by the presence of extended loop motifs that form the active site. In different structures of the bacterial enzymes, these loops adopt different conformations, and appear to play a role in recognizing the extended structure of the N-glycan substrate.  Binary complexes with two inhibitors, &alpha;-glucosyl-1,3-deoxymannonojirimycin and &alpha;-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-&alpha;-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in ''Bx''GH99; Glu332 in ''Bt''GH99) and another key residue that interacts with both the 2OH of the -1 mannose residue and the 6OH of the -2 glucose residue, which provides a rationale for the requirement of a glucosylated-mannoside as the minimal substrate for GH99 enzymes <cite>Thompson2012</cite>. As discussed in more detail in the "Kinetics and Mechanism" section, there is not nucleophilic residue within potential bonding distance to the anomeric centre, consistent with the neighboring group participation mechanism via an epoxide intermediate.
 Comparison of binary complexes of &alpha;-mannosyl-1,3-isofagomine and &alpha;-glucosyl-1,3-isofagomine with ''B. xylanisolvens'' GH99 revealed almost identical binding <cite>#Hakki2014</cite>. It was speculated that a key residue in the -2 subsite, tryptophan-126 in ''Bx''GH99, provided a preference for mannose-configured substrates in these bacterial endo-α-1,2-mannanases. By contrast the equivalent residue in mammalian endo-α-mannosidases is a highly conserved tyrosine, which it was speculated may result in a favorable hydrogen bond to a glucose-configured substrate and confer endo-α-mannosidase activity.
-The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with ad without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020</cite>.
+The catalytic domain of human MANEA and complexes with substrate-derived inhibitors were consistent with 3D structures of equivalent complexes with the ''Bacteroides'' enzymes. , which provide insight into dynamic loop movements that occur on substrate binding. MANEA contains a flexible '-2' loop (which is absent in bacterial enzymes), which interacts with the -2 sugar. In various structures of MANEA, with and without ligands bound, the -2 loop was observed to adopt different conformations <cite>Sobala2020P</cite>.
 which was observed
 A GH99-like protein from the marine flavobacterium ''Ochrovirga pacifica'', which lacks the conserved catalytic residues of glycoside hydrolase family 99 possessed a similar fold to other GH99 members. It is speculated that this protein may be noncatalytic and possibly involved in carbohydrate binding <cite>Robb2017</cite>.

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 ;First sterochemistry determination: ''Bacteroides thetaiotaomicron'' ''endo''-&alpha;-mannosidase by <sup>1</sup>H NMR <cite>Thompson2012</cite>
-;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2021</cite>
+;First [[catalytic nucleophile]] identification: GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue <cite>Thompson2012 Sobala2020A</cite>
 ;First [[general acid/base]] residue identification: ''Bacteroides thetaiotaomicron'' ''endo''-&alpha;-mannosidase by X-ray crystallography and analysis of enzyme mutant activities <cite>Thompson2012</cite>

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 #Hakki2014 pmid=25487964
 #Sobala2020A pmid=32490192
-#Sobala2021P pmid=33154157
+#Sobala2020P pmid=33154157
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH099]]