The Structure of an Archaeal α-Glucosaminidase Provides Insight into Glycoside Hydrolase Evolution

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.

A Novel Dimeric Exoglucanase (GH5_38): Biochemical and Structural Characterisation towards its Application in Alkyl Cellobioside Synthesis

“…The dimer displayed a higher activity of about two activity units (compared to the monomer) on the tested substrates, except for pachyman and curdlan. The active monomer of the Exg-D enzyme was found to be novel, as several studies have generally shown that dimeric oligomers of the GH enzymes are active, while their monomers are not active or display very low activity [13,[15][16][17]. The specific activity results were supported by biochemical characterisation and kinetic studies, which showed that the dimeric and monomeric species of Exg-D displayed the same pH and temperature optima.…”

Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

“…From the structural comparison, Glu179 and Glu347 of GlmA Tk are sterically identical to the acid/base Glu188 and the nucleophile Glu268 of Hs-β-gal, respectively (Figure 4A, B, C). GlmA Tk mutations, E179Q and E347Q, resulted in dramatic activity loss [15], supporting the notion that these residues are involved in protein catalysis. Furthermore, these Glu residues are located in the β4 and β7 strands of the TIM-barrel domain and are separated by 4.8 Å [15].…”

“…GlmA Tk mutations, E179Q and E347Q, resulted in dramatic activity loss [15], supporting the notion that these residues are involved in protein catalysis. Furthermore, these Glu residues are located in the β4 and β7 strands of the TIM-barrel domain and are separated by 4.8 Å [15]. All proteins in the GH35 family belong to a GH-A clan that comprises enzymes with two conserved catalytic Glu residues in the C-terminals of β4 and β7 [17].…”

“…The structure of GlmA Ph was deduced using the single-wavelength anomalous dispersion of selenomethionine atoms and refined at 2.60-Å resolution (PDB 5GSL) [15]. The structure of GlmA Pf and GlcN-bound GlmA Tk was determined at 1.75-Å resolution (PDB 6JOW, unpublished) and 1.27-Å resolution (PDB 5GSM) [15], respectively, using molecular replacement of the structure of the GlmA Ph monomer as the search model. The structures of GlmA Ph and GlmA Pf showed little variation to that of GlmA Tk , as reflected in the RMSD values of 0.90 Å for 767 Cα atoms and 0.74 Å for 751 Cα atoms, respectively (Figure 1A).…”

“…To address this critical question, we determined the crystal structure of GlmA Tk (encoded by the TK1754 gene) from Thermococcus kodakaraensis KOD1 [15]. The crystal structures of two proteins, which are highly homologous to GlmA Tk , GlmA Ph (encoded by the PH0511 gene) [16] and GlmA Pf (encoded by the PF0363 gene) [14], from the closely related hyperthermophiles Pyrococcus species Pyrococcus horikoshii and Pyrococcus furiosus , respectively, were also determined.…”