Golgi alpha-mannosidase II cleaves two sugars sequentially in the same catalytic site

Abstract

Golgi alpha-mannosidase II (GMII) is a key glycosyl hydrolase in the N-linked glycosylation pathway. It catalyzes the removal of two different mannosyl linkages of GlcNAcMan(5)GlcNAc(2), which is the committed step in complex N-glycan synthesis. Inhibition of this enzyme has shown promise in certain cancers in both laboratory and clinical settings. Here we present the high-resolution crystal structure of a nucleophile mutant of Drosophila melanogaster GMII (dGMII) bound to its natural oligosaccharide substrate and an oligosaccharide precursor as well as the structure of the unliganded mutant. These structures allow us to identify three sugar-binding subsites within the larger active site cleft. Our results allow for the formulation of the complete catalytic process of dGMII, which involves a specific order of bond cleavage, and a major substrate rearrangement in the active site. This process is likely conserved for all GMII enzymes-but not in the structurally related lysosomal mannosidase-and will form the basis for the design of specific inhibitors against GMII.

Fig. 1.

Golgi α-mannosidase II–substrate interactions. (A) Golgi α-mannosidase II catalyzes the cleavage of two mannosyl linkages, an α1,3-linkage between M3 and M4 and an α1,6-linkage between M3 and M5, converting GnMan₅Gn₂ to GnMan₃Gn₂. (B) GnMan₅Gn fitted to the F_o − F_c electron density in the dGMII active site. The electron density is contoured to 3.0 σ. The crystallographic temperature factors represent the average values for the carbon and oxygen atoms of the saccharide. (C) dGMII–GnMan₅Gn interactions as determined by HBPLUS/LIGPLOT.

Fig. 2.

GMII–substrate. GnMan₅Gn (yellow) is bound in the dGMII(D204A) active site (white). Hydrogen bonds are depicted as green dashed lines, and a single zinc ion is presented in purple and aids to coordinate M5 in the catalytic site.

Fig. 3.

Comparison of GnMan₅Gn and Man₅ bound to dGMII. The absence of the G3 anchor position in Man₅ (green) results in a very different mode of binding, particularly distal from the catalytic and holding sites. The saccharide shifts are 11 Å for M1 and 5 Å for M2.

Fig. 4.

The M5 saccharide compared with a covalent reaction intermediate and a synthetic tetrasaccharide. (A) Position M5 in the catalytic site (yellow) superimposed on 5-fluorogulosyl fluoride (slate), which is covalently attached (blue dashed line) to OE2 of Asp-204. A zinc ion is in shown in purple. The distance between the C1 positions of each saccharide moiety is ≈2 Å. Such a distance could be traversed by a distortion from a ⁴C₁ to a ¹S₅—which is what is proposed to occur concurrent with nucleophilic attack by Asp-204. (B) A superposition of the mannosyl moieties in the catalytic site of dGMII from the full substrate (yellow) and a synthetic tetrasaccharide (green).

Fig. 5.

Comparison of the dGMII and bLM active site clefts. (A) A surface model of dGMII depicting the substrate-binding site and colored according to a multiple sequence alignment. More intense red shading indicate increased sequence conservation. Note the high degree of conservation in the cleft where the substrate (yellow) binds. The catalytic (M5), holding (M4), and anchor (G3) sites are indicated. (B) The bLM active site aligned in the same orientation as A above. The dGMII substrate is indicated as partially transparent. There is a lack of sequence conservation at both the holding and the anchor sites and an increased width of the active site cleft between dGMII and bLM.

Fig. 6.

A proposed schematic of the GMII reaction. The reaction begins with the binding of the GnMan₅Gn₂ oligosaccharide to GMII, with the M5 position binding in the catalytic site, M4 occupying the holding site, and G3 in the anchor site (1). After the hydrolysis of M5 by the enzyme, the catalytic site is free (2). Oligosaccharide rearrangement ensues to allow M4 to enter the catalytic site (3). M4 is subsequently hydrolyzed in the catalytic site by a similar mechanism, and the M4 release is concurrent with product release (4).