Structure and Kinetic Investigation of Streptococcus pyogenes Family GH38 α-Mannosidase

Abstract

BackgroundThe enzymatic hydrolysis of α−mannosides is catalyzed by glycoside hydrolases (GH), termed α−mannosidases. These enzymes are found in different GH sequence–based families. Considerable research has probed the role of higher eukaryotic “GH38” α−mannosides that play a key role in the modification and diversification of hybrid N-glycans; processes with strong cellular links to cancer and autoimmune disease. The most extensively studied of these enzymes is the Drosophila GH38 α−mannosidase II, which has been shown to be a retaining α−mannosidase that targets both α−1,3 and α−1,6 mannosyl linkages, an activity that enables the enzyme to process GlcNAc(Man)5(GlcNAc)2 hybrid N-glycans to GlcNAc(Man)3(GlcNAc)2. Far less well understood is the observation that many bacterial species, predominantly but not exclusively pathogens and symbionts, also possess putative GH38 α−mannosidases whose activity and specificity is unknown.Methodology/Principal FindingsHere we show that the Streptococcus pyogenes (M1 GAS SF370) GH38 enzyme (Spy1604; hereafter SpGH38) is an α−mannosidase with specificity for α−1,3 mannosidic linkages. The 3D X-ray structure of SpGH38, obtained in native form at 1.9 Å resolution and in complex with the inhibitor swainsonine (K i 18 µM) at 2.6 Å, reveals a canonical GH38 five-domain structure in which the catalytic “–1” subsite shows high similarity with the Drosophila enzyme, including the catalytic Zn2+ ion. In contrast, the “leaving group” subsites of SpGH38 display considerable differences to the higher eukaryotic GH38s; features that contribute to their apparent specificity.Conclusions/SignificanceAlthough the in vivo function of this streptococcal GH38 α−mannosidase remains unknown, it is shown to be an α−mannosidase active on N-glycans. SpGH38 lies on an operon that also contains the GH84 hexosaminidase (Spy1600) and an additional putative glycosidase. The activity of SpGH38, together with its genomic context, strongly hints at a function in the degradation of host N- or possibly O-glycans. The absence of any classical signal peptide further suggests that SpGH38 may be intracellular, perhaps functioning in the subsequent degradation of extracellular host glycans following their initial digestion by secreted glycosidases.

Analysis of a New Family of Widely Distributed Metal-independent α-Mannosidases Provides Unique Insight into the Processing of N-Linked Glycans

“…Mannosidases in families GH38, GH47, and GH92, which are structurally and mechanistically distinct and hydrolyze a variety of ␣-mannosidic linkages, all require the coordination of a divalent metal ion in the catalytic site for activity. The divalent metal ion in the active site is thought to aid in distorting the substrate and stabilize the transition state by bridging the O2 and O3 hydroxyl groups of the mannoside bound in the Ϫ1 subsite (3,4,35). The absence of a participating metal ion in the GH125 mechanism reveals that divalent cation involvement is not a general requirement for efficient hydrolysis of ␣-mannosides.…”

“…␣-Mannosidases known to process N-glycans are found in families 38, 47, 76, 92, and 99. Very recent studies have shown the bacterial family 38 ␣-mannosidase from Streptococcus pyogenes (SpyGH38) to be a specific exo-␣1,3-mannosidase that is tolerant of the ␣1,6-branches in N-glycans (3). Analysis of family 92 glycoside hydrolases from the human gut symbiont Bacteroides thetaiotaomicron revealed an expanded repertoire of ␣-mannosidases (4).…”

The N-Glycan Cluster from Xanthomonas campestris pv. campestris

“…More recently, enzymes from Streptococcus pyogenes and Streptococcus pneumoniae classified in the GH38 (␣-1,3-mannosidase) and GH125 (␣-1,6-mannosidase) families, respectively, have been identified. These enzymes are active on N-glycans, highlighting the processing of N-glycans by Streptococcus bacteria (18,19). Finally, it was shown that the human pathogen Capnocytophaga canimorsus deglycosylates surface glycoproteins from the host and supports its growth on the released glycan moiety (20).…”

Mutational Analysis of Divalent Metal Ion Binding in the Active Site of Class II α-Mannosidase from Sulfolobus solfataricus

“…1 In the absence of substrate in the active site, the coordination sphere of the bound metal ion is T 5 -square-based pyramidal with the four mentioned side chains and a single water molecule as ligands. 9,15 Of the two aspartyls complexing the metal ion, one is also the nucleophile in the otherwise classical catalytic machinery of the glycosidase retaining mechanism. 1 The divalent metal ion bound to the active site is a prerequisite for binding of the substrate.…”