Glycoside Hydrolase Family 99

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Glycoside Hydrolase Family GH99
Clan	not assigned
Mechanism	retaining
Active site residues	known
CAZy DB link
https://www.cazy.org/GH99.html

Substrate specificities

Figure 1. (Top) Endo-α-mannosidase is located in the Golgi apparatus and pre-Golgi intermediates and acts on folded and unfolded ER-escaped glucosylated N-linked glycoproteins (X = protein); glucosylated free oligosaccharides (X = OH); and dolichol-bound glucosylated glycans (X = diphosphodolichol). (Bottom) Endo-α-1,2-mannanases are bacterial enzymes with the ability to cleave Manα1-3Manα1-2Manα1-2Manα substructures within fungal cell wall mannan at the indicated positions, releasing α-1,3-mannobiose.

Glycoside hydrolases of family GH99 are endo-acting enzymes. This family was originally created from the mammalian enzyme, MANEA, cloned by Spiro and co-workers [1]. Two main activities have been described for this family. Endo-α-1,2-mannosidase activity describes the capacity to cleave glucose-substituted mannose within immature N-linked glycans of the general formula Glc_1-3Man₉GlcNAc₂ (or structures trimmed in the B and C branches). On the other hand endo-α-1,2-mannanase activity describes the ability to cleave αMan-1,3-αMan-1,2-αMan-1,2-αMan sequences within fungal cell wall mannans, releasing α-1,3-mannobiose [2].

Mammalian endo-α-mannosidases typically possess maximal activity on the monoglucosylated forms [3]. Mammalian endo-α-mannosidases are localized to the Golgi apparatus [4] and appear to play a role in the rescue of glucosylated N-linked glycans that have evaded the action of the endoplasmic reticulum exo-glucosidases I and II [5]. Mammalian endo-α-mannosidase has greater activity on glucosylated N-linked glycans that have been trimmed in the non-glucose-substituted branches [1]. There is evidence that mammalian endo-α-mannosidases act on dolichol-bound N-glycan precursors [6], as well as free oligosaccharides released from N-glycoproteins and which undergo retrograde transport through the secretory pathway [7]. A fluorescent tetrasaccharide fragment (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) has been developed that is a substrate for mammalian endo-α-mannosidase [8]. Studies of bacterial GH99 enzymes from Shewanella amazonensis [9], Bacteroides thetaiotaomicron and Bacteroides xylanisolvens [10] have shown that these enzymes can also process Glc_1/3Man_9/7GlcNAc₂ structures, matching the substrate specificity of the mammalian enzymes. Kinetic analysis of Bacteroides thetaiotaomicron GH99 on a fluorescently-labelled Glc₃Man₉GlcNAc₂ structure yielded kinetic parameters that were similar to that found for the mammalian enzyme [10]. Both mammalian and bacterial enzymes can utilize simple 'reducing end' substrate mimics such as methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside [11] or α-glucosyl-1,3-α-mannopyranosyl fluoride [10], but are inactive on aryl mannosides.

Bacterial GH99 enzymes from B. thetaiotaomicron and B. xylanisolvens have superior kinetics for cleavage of Manα1-3Manα1-2Manα1-2Manα-OMe versus glucosylated high mannose N-glycans and are thus more appropriately considered endo-α-1,2-mannanases [2]. These two enzymes displayed a 10-fold preference for methylumbelliferyl α-mannosyl-1,3-α-mannopyranoside versus methylumbelliferyl α-glucosyl-1,3-α-mannopyranoside [2].

Kinetics and Mechanism

Family GH99 endo-α-mannosidases are retaining enzymes, as first shown by ¹H NMR analysis of the hydrolysis of α-glucosyl-1,3-α-mannopyranosyl fluoride by Bacteroides thetaiotaomicron [10]. Retaining mannoside hydrolases (eg GH38) typically follow a classical Koshland double-displacement mechanism. However, X-ray crystallographic analysis of BxGH99 and BtGH99 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 [10]. In this proposal, Glu333 in BxGH99 (Glu329 in BtGH99) 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_cat/K_M for anomeric-²H and anomeric-¹³C support an oxocarbenium ion-like transition state, and that for C2-¹⁸O (1.052 ± 0.006) implicates nucleophilic participation by the C2-oxygen [12]. Additional support through quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar [12].

Figure 2. Proposed catalytic mechanisms for GH99 endo-α-mannosidase. (A) Classical Koshland double-displacement mechanism. (B) Neighboring group participation via a 1,2-anhydrosugar intermediate.

Catalytic Residues

The general acid/base was highlighted by X-ray crystallographic analysis as Glu336 in BxGH99 and Glu332 in BtGH99 [10]. The Glu332Ala mutant of BtGH99 shows a 50-fold decrease in catalytic activity relative to the wild-type enzyme using the activated substrate α-glucosyl-1,3-α-mannopyranosyl fluoride, and undetectable activity against the natural substrate, Glc₃Man₇GlcNAc₂, 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 [10]. According to this proposal, Glu333 in BxGH99 (Glu329 in BtGH99) acts as a general acid/base to deprotonate the 2OH and protonate the 1,2-anhydrosugar.

Three-dimensional structures

Three-dimensional structures are available for bacterial members of GH99, including B. thetaiotaomicron and B. xylanisolvens [10]. They have a classical (β/α)₈ 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, α-glucosyl-1,3-deoxymannonojirimycin and α-glucosyl-1,3-isofagomine, and 'active-site-spanning' ternary complexes with the same two inhibitors and the reducing end product fragment 1,2-α-mannobiose, provided insight into active site residues, especially the acid/base (Glu336 in BxGH99; Glu332 in BtGH99) 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 [10]. As discussed in more detail in the "Kinetics and Mechanism" section, the precise identity of the nucleophilic residue is unclear, as in all GH99-inhibitor complexes with an occupied -1 subsite there is no candidate nucleophile within a reasonable distance to the "anomeric" carbon: in BxGH99 Glu333 is approximately 3.5 Å distant and the OH of Tyr46 and Tyr252 are 4.0 Å distant.

Comparison of binary complexes of α-mannosyl-1,3-isofagomine and α-glucosyl-1,3-isofagomine with B. xylanisolvens GH99 revealed almost identical binding [2]. It was speculated that a key residue in the -2 subsite, tryptophan-126 in BxGH99, 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.

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 [13].

Family Firsts

First sterochemistry determination: Bacteroides thetaiotaomicron endo-α-mannosidase by ¹H NMR [10]

First catalytic nucleophile identification: It has been proposed that GH99 enzymes operate through a mechanism involving substrate assisted catalysis by the 2OH group of the -1 mannose residue [10]

First general acid/base residue identification: Bacteroides thetaiotaomicron endo-α-mannosidase by X-ray crystallography and analysis of enzyme mutant activities [10]

First 3-D structure of a GH99 enzyme: Bacteroides thetaiotaomicron and Bacteroides xylanisolvens endo-α-mannosidases [10]

References

All Medline abstracts: PubMed