Glycoside Hydrolase Family 109

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Glycoside Hydrolase Family GH109
Clan	none
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/GH109.html

Substrate specificities

The only activity so far identified in this recently discovered family of glycoside hydrolases is that of α-N-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from Elizabethkingia meningosepticum. Because it operates at neutral pH optimum, this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O [1]. The enzyme clearly prefers GalNAc over Gal as the aglycon as indicated by a 2,000-fold reduction in kcat for the hydrolysis of p-nitrophenyl α-galactoside compared with p-nitrophenyl α-N-acetyl-galactosaminide and by a more than tenfold increase in K_m [1].

Kinetics and Mechanism

Family GH109 enzymes operate via the unusual NAD-dependent hydrolysis mechanism involving an NAD⁺ cofactor, that so far has been seen only in Glycoside Hydrolase Family 4 (GH4), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by α-N-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar retaining mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated intermediate. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD⁺ state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases [1].

Catalytic Residues

A stated above the enzymes of this family do not use a classical acid/base catalysis, but instead use a rare catalytic mechanism involving NAD-dependent hydrolysis with an NAD+ cofactor, highly similar to that seen in family Glycoside Hydrolase Family 4. The catalytic machinery therefore comprises NAD⁺ and Tyr-179, which abstracts H-2 to form the unsaturated intermediate. Subtle differences with family GH4 exist, however, such as the absence in GH109 of an identifiable acid to assist glycosidic bond cleavage. It is believed that this enables the hydrolysis of the 'wrong' anomer.

Three-dimensional structures

The three-dimensional structure of Elizabethkingia meningosepticum α-N-acetylgalactosaminidase has been reported in 2007 [1]. The closest structural relatives belong to the Gfo/Idh/MocA oxidoreductase family (Z-score of 29.4 and r.m.s. deviation of 3.0 A for 329 equivalent Ca-atoms for Zymomonas mobilis glucose-fructose oxidoreductase (PDB 1ofg). More distant structural homologs are identified by means of the classical Rossmann fold. The structural similarity includes the active-site architecture, where the spatial arrangement of NAD+ and several other residues is conserved, suggesting a common ancestor that has evolved its NAD+-based molecular mechanism to adapt to diverse metabolic requirements [1].

Family Firsts

First sterochemistry determination: Elizabethkingia meningosepticum α-N-acetylgalactosaminidase by ¹H NMR [1].
First mechanistic identification: Elizabethkingia meningosepticum α-N-acetylgalactosaminidase, by deuterium exchange of H-2, involvement of NAD⁺ and structural similarity with GH4 enzymes [1].
First 3-D structure: Elizabethkingia meningosepticum α-N-acetylgalactosaminidase [1] (PDB 2ixa) and (PDB 2ixb).

References

Liu QP, Sulzenbacher G, Yuan H, Bennett EP, Pietz G, Saunders K, Spence J, Nudelman E, Levery SB, White T, Neveu JM, Lane WS, Bourne Y, Olsson ML, Henrissat B, and Clausen H. (2007). Bacterial glycosidases for the production of universal red blood cells. Nat Biotechnol. 2007;25(4):454-64. DOI:10.1038/nbt1298 | PubMed ID:17401360 [1]