Abstract
The archaeal exo-β-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 β-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 β-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 β-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the "evolutionary heritage residue" it shares with GH35 β-galactosidase. Our study suggests that GH35 and GH42 β-galactosidases evolved by exploiting the structural features of GlmA.
Highlights
The archaeal exo--D-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines
GlmA can differentiate glucosamine from galactose based on one charged residue while using the “evolutionary heritage residue” it shares with GH35 -galactosidase
Our analysis showed that a large surface area of 5530 Å2 is buried in the structure and that 29 hydrogen bonds and 16 salt bridges are created upon dimer formation. These findings indicate that both monomers are intimately associated, which might contribute to the high thermostability of GlmATk
Summary
Overall Structure—The structure of GlmAPh was solved using the single-wavelength anomalous dispersion of selenomethionine atoms and refined at 2.6 Å resolution (Table 1). Despite low sequence similarities (15–17%), the three domain structures of monomer GlmATk could be readily superimposed on four GH42 -galactosidases with Z scores Ͼ25 and RMSD values of 2.6 –3.0 Å for equivalent C␣ atoms, except for 80 residues of the C-terminal region of GlmATk (Fig. 2A). The superimposition of the TIM-barrel domain of GlmATk with Tri--gal revealed a high degree of structural similarity between the Ϫ1 subsites of these proteins (Fig. 4B), their substrates are different Both the GlcN and galactose molecules adopt a chair conformation with their C1 hydroxyl group (O1) in the -anomer configuration. The Discrimination of GlcN from Galactose by GlmATk— Despite the high structural similarities at the active site, radical differences were observed in the rest of the substrate-binding residues of GlmATk: Asp178, Tyr379, and Glu306. The necessity of this interaction is supported by a previous report indicating that
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