Structure–function relationships in Gan42B, an intracellular GH42 β-galactosidase fromGeobacillus stearothermophilus

Abstract

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a battery of degrading enzymes for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. A 9.4 kb gene cluster has recently been characterized in G. stearothermophilus that encodes a number of galactan-utilization elements. A key enzyme of this degradation system is Gan42B, an intracellular GH42 β-galactosidase capable of hydrolyzing short β-1,4-galactosaccharides into galactose units, making it of high potential for various biotechnological applications. The Gan42B monomer is made up of 686 amino acids, and based on sequence homology it was suggested that Glu323 is the catalytic nucleophile and Glu159 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Gan42B (at 2.45 Å resolution) and its catalytic mutant E323A (at 2.50 Å resolution), as determined by X-ray crystallography, are reported. These structures demonstrate that the three-dimensional structure of the Gan42B monomer generally correlates with the overall fold observed for GH42 proteins, consisting of three main domains: an N-terminal TIM-barrel domain, a smaller mixed α/β domain, and the smallest all-β domain at the C-terminus. The two catalytic residues are located in the TIM-barrel domain in a pocket-like active site such that their carboxylic functional groups are about 5.3 Å from each other, consistent with a retaining mechanism. The crystal structure demonstrates that Gan42B is a homotrimer, resembling a flowerpot in general shape, in which each monomer interacts with the other two to form a cone-shaped tunnel cavity in the centre. The cavity is ∼35 Å at the wide opening and ∼5 Å at the small opening and ∼40 Å in length. The active sites are situated at the interfaces between the monomers, so that every two neighbouring monomers participate in the formation of each of the three active sites of the trimer. They are located near the small opening of the cone tunnel, all facing the centre of the cavity. The biological relevance of this trimeric structure is supported by independent results obtained from gel-permeation chromatography. These data and their comparison to the structural data of related GH42 enzymes are used for a more general discussion concerning structure-activity aspects in this GH family.

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

“…The active sites of glycoside hydrolases are classified into three types: cleft type, tunnel type, and pocket type (23). Both GlmA and GH42 ␤-galactosidases have a cleft-type active site in their monomeric forms; however, the shape of the active site changes to a pocket type upon oligomerization, which can better accommodate smaller substrates (24) (Fig. 6).…”

“…A, superposition of the trimer forms of GH42 ␤-galactosidases, A4-␤-gal (light yellow), Bca-␤-gal (cyan), Gan42B (light blue), and BlGal42A (light green). Comparing the structure of Gan42B with the other three structures revealed high structural similarities, with RMSD values of 1.2-2.0 Å(24). The right panel shows the dimer forms of GlmA Tk .…”

Discovery of α-l-arabinopyranosidases from human gut microbiome expands the diversity within glycoside hydrolase family 42

“…3A). Superimposing the docked paeonolide from BlArap42B into BlGal42A shows the phenyl aglycone clashes with the backbone of Trp 332 , which is invariant and similarly spatially located in structurally characterized GH42 enzymes (13,18,22,26,27), and Phe 226 of the neighboring monomer in BlGal42A, which can explain its lack of activity for paeonolide (Fig. 3C).…”

“…The residues creating the spatial and chemical environment at subsite Ϫ1 are invariant in characterized GH42 ␤-galactosidases (13,18,22,26,27). However, the change from a histidine residue in classical GH42 ␤-galactosidases to Trp 358 in BlArap42B limits the space at subsite Ϫ1, which would cause clashing with C6 of galactose in accordance with BlArap42B being unable to hydrolyze ␤-galactosides (Fig.…”

Structural basis for enzyme bifunctionality – the case of Gan1D from Geobacillus stearothermophilus

“…We have previously characterized a specific galactanutilization gene cluster in G. stearothermophilus T-1 [31]. The 9.4 kb cluster encodes, among other proteins, for an extracellular galactanase (Gan53A), an intracellular galactosidase (Gan42B) [56], and a specific ABC sugar transporter (GanEFG) [31]. Unexpectedly, we also identified another 12.5 kb gene cluster that, based on real-time RT-PCR analysis, is induced by galactan and galactose, suggesting that this cluster is also involved in the utilization of galacto-saccharides [57].…”