A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase fromGeobacillus stearothermophilus

Abstract

Geobacillus stearothermophilus T6 is a thermophilic, Gram-positive soil bacterium that possesses an extensive and highly regulated hemicellulolytic system, allowing the bacterium to efficiently degrade high-molecular-weight polysaccharides such as xylan, arabinan and galactan. As part of the xylan-degradation system, the bacterium uses a number of side-chain-cleaving enzymes, one of which is Axe2, a 219-amino-acid intracellular serine acetylxylan esterase that removes acetyl side groups from xylooligosaccharides. Bioinformatic analyses suggest that Axe2 belongs to the lipase GDSL family and represents a new family of carbohydrate esterases. In the current study, the detailed three-dimensional structure of Axe2 is reported, as determined by X-ray crystallography. The structure of the selenomethionine derivative Axe2-Se was initially determined by single-wavelength anomalous diffraction techniques at 1.70 Å resolution and was used for the structure determination of wild-type Axe2 (Axe2-WT) and the catalytic mutant Axe2-S15A at 1.85 and 1.90 Å resolution, respectively. These structures demonstrate that the three-dimensional structure of the Axe2 monomer generally corresponds to the SGNH hydrolase fold, consisting of five central parallel β-sheets flanked by two layers of helices (eight α-helices and five 310-helices). The catalytic triad residues, Ser15, His194 and Asp191, are lined up along a substrate channel situated on the concave surface of the monomer. Interestingly, the Axe2 monomers are assembled as a `doughnut-shaped' homo-octamer, presenting a unique quaternary structure built of two staggered tetrameric rings. The eight active sites are organized in four closely situated pairs, which face the relatively wide internal cavity. The biological relevance of this octameric structure is supported by independent results obtained from gel-filtration, TEM and SAXS experiments. These data and their comparison to the structural data of related hydrolases are used for a more general discussion focusing on the structure-function relationships of enzymes of this category.

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

“…The structural homology with the other members of SGNH hydrolase family proteins suggests that residues involved in oxyanion hole formation of Gly63 and Asn92 (Alalouf et al 2011). The crystal structures of AcXE2 also confirms the arrangement of these residue to maintain exact distance and appropriate orientation for interacting and stabilising the catalytic anionic intermediate (Lansky et al 2014). The oligomeric catalytic site arrangement in AcXE2 deviates from the typical serine esterases, although the active site of each AcXE2 monomer follows as typical serine esterases.…”

“…Structural and functional properties of monomeric and octameric Geobacillus stearothermophilus AcXE were revealed by Lansky et al (2014). The AcXE2 complete protein structure resembles to the SGNH hydrolase fold and principally similar to α/β hydrolase fold differentiated by the location of the catalytic residues and the lack of nucleophilic elbow (Wei et al 1995).…”

“…The five-central parallel-β-sheet edged by two layers of helices can be classified into eight α-helices and five 3 10 helices, altogether constituting to the fold. The positions of eight β-helices, five helices and 3 10 helices are as follows: 8–13 (β1), 55–58 (β2), 84–88 (β3), 130–134 (β4), 168–170 (β5), 14–19 (α1), 36–49 (α2), 65–77 (α3), 90–100 (α4), 109–125 (α5), 145–166 (α6), 171–181 (α7) and 196–211 (α8) and 50–53 (h1), 78–81 (h2), 103–107 (h3), 126–128 (h4), and 184–188 (h5), respectively (Lansky et al 2014). The structural positions of these eight β-helices display a topology of −1x + 2x + 1x + 1x, structurally this monomer can be divided into two parts convex and concave surfaces.…”

“…Four salt bridges between Glu105 OE1 and OE2 of one chain and Arg55 NH1 and NH2 of the other chain maintain the contact between the two tetramers. AcXE2 octamer contains eight polypeptide chains located around the central cavity, which is sectioned into four local hollows; these hollows are highly significant due to the presence of two active sites (belonging to different chains and different tetramers) separated by 5 Å, approximately (Lansky et al 2014). Thus, AcXE2 enzyme contains four pairs of double catalytic sites located around the C4 axis of the octamer facing towards the central cavity.…”

“…Experiments conducted by Alalouf et al (2011) have also confirmed the catalytic function of AcXE2 on 2,3,4-tri-O-acetyl methyl-β-D-xylopyranoside, where AcXE2 was able to deacetylate at positions 3 and 4 simultaneously (Alalouf et al 2011). The octameric arrangement of AcXE2 is greatly stabilised by intermolecular interactions when compared to dimeric, tetrameric structures, thus enhancing the thermodynamic stability of the multimeric assembly (Lansky et al 2014) (Figure 7) (Table 3). …”