Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

Abstract

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1–22), an N-terminal region (NTR; residues 23–135) and a catalytic domain (residues 136–324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23–324) was successfully determined. The crystal diffracted to 1.9 Å resolution and belonged to space group I212121. The catalytic domain (residues 136–321) exhibited a (β/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23–135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Functional analysis of the N‐terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis

“…The symmetric interactions at interfaces II and III promoted the rapid assembly of dimers to form crystals. Although our previous report could not clearly refer to the presence of NTR in the crystals, the present results suggest that NTR was cleaved over a long crystallization time (6 months) and then assembled and crystallized through electrostatic interactions [10]. These results suggest that the NTR functions in way to cover the molecular surface of the catalytic domain and prevent dimerization, causing the rapid formation of protein crystals.…”

“…The results showed that TTE0866 was disordered from Leu47 to Asp94 located on the NTR, which matched well with the results obtained in PONDR. The residues 23-46 were confirmed to be a highly hydrophilic region similar to the residues 47-94 by the hydropathy plot described in the previous study [10]. Thus, the majority of the NTR (72 of 113 residues, 64%) is a disordered region.…”

“…E. Rosetta2 (DE3) pLysS cells (Novagen) harboring the expression plasmid were cultured in Luria‐Bertani (LB) medium, and expression of the gene was induced by isopropyl β‐D‐1‐thiogalactopyranoside (IPTG). Purification of WT, TTE Δ20 , TTE Δ40 , TTE Δ52 , and TTE Δ60 was performed as previously described [ 10 ]. Purification of TTE Δ80 , TTE Δ100 , and TTE Δ110 was performed by a slightly modified procedure using hydrophobic interaction chromatography (HIC) with a HiTrap Butyl HP column (Cytiva, Tokyo, Japan) instead of anion‐exchange chromatography (AEX).…”

“…The amino acid number of TTE0866 described in this report corresponds to the protein including the signal sequence. We previously determined the crystal structure of wild‐type TTE0866 (WT), containing residues 23–324 (the NTR and CE4 domains), to explain its high activity toward CA with a high DS [ 10 ]. Although the active site conformation of TTE0866 was highly similar to that of other CE4 enzymes, the orientation of the tryptophan residue (W264) near the active site was clearly distinct.…”

“…In this study, we investigated the functional role of the NTR of TTE0866. The NTR of TTE0866 has several characteristics, as suggested by sequence analysis [ 10 ]. The NTR is mostly comprised of hydrophilic and charged amino acid residues.…”