Structure–specificity relationships in Abp, a GH27 β-L-arabinopyranosidase fromGeobacillus stearothermophilusT6

Abstract

L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer made up of two `open-pincers' dimers, which clamp around each other to form a central cavity. The four active sites of the Abp tetramer are situated on the inner surface of this cavity, all opening into the central space of the cavity. The biological relevance of this tetrameric structure is supported by independent results obtained from size-exclusion chromatography (SEC), dynamic light-scattering (DLS) and small-angle X-ray scattering (SAXS) experiments. These data and their comparison to the structural data of related GH27 enzymes are used for a more general discussion concerning structure-selectivity aspects in this glycoside hydrolase (GH) family.

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

“…In addition to these multiple hydroxyl hydrogen bonds, several aromatic -stacking interactions with the bound galactose are formed by Phe54, Tyr287, Trp331, Phe361 and Trp197 (Fig. 4c), enzyme-ligand interactions of the types that are commonly observed amongst glycoside hydrolases (Solomon et al, 2007;Lansky, Salama, Solomon et al, 2014). As mentioned above, the highly conserved Trp197 belongs to the adjacent symmetry-related monomer and seems to complement the active-site pocket of Gan42B (Fig.…”

“…The three active sites of Gan42B are located near the small opening of the cone tunnel (10-15 Å from the bottom), all facing the centre of the trimer (Fig. 3a) observed in other glycoside hydrolases (Lansky, Salama, Solomon et al, 2014). The Gan42B trimer is held together by a number of hydrogen bonds, salt bridges and -stacking interactions (Table 3).…”

“…Although various oligomeric assemblies are increasingly being observed and reported in GH structures (Lansky, Alalouf, Solomon et al, 2014;Lansky, Salama, Solomon et al, 2014), the reasons for such oligomerizations are not always completely clear. In the particular case of Gan42B presented here, the specific oligomeric assembly observed is obviously driven primarily by the catalytic activity, as the full active sites are formed in the monomer-monomer interfaces created by such an assembly.…”

“…The residues that interact directly with this bound glycerol are Glu371, Asn158, Arg120, Phe54 and Phe361, the catalytic residues Glu323 and Glu159, and Asp21, which forms a water-mediated interaction with the O2 atom of the activesite glycerol. Since glycerol often mimics partial structures of sugars owing to similarly positioned chemical functional groups (Solomon et al, 2007;Lansky, Salama, Solomon et al, 2014), these residues are probably also involved in interaction with real substrates of Gan42B, especially considering their The active site of Gan42B. (a) An enlargement of the H-A domain interface, demonstrating how subdomain H (orange, right) complements the active site situated mainly in domain A (red, left).…”

Crystal Structure and Mutational Analysis of Isomalto-dextranase, a Member of Glycoside Hydrolase Family 27

“…Domain A shows a significant similarity to the domains of GH27, GH36, GH31, and GH13 enzymes, all of which consist of (␤/␣) 8 -barrel folds (Table 2). Among the GH27 enzymes, similarities with ␤-L-arabinopyranosidases (25,29) were relatively higher than those with ␣-N-acetylgalactosaminidases and ␣-galactosidases (30). Domain C is similar to the antiparallel ␤-sheet domains of various GH family enzymes.…”