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

Abstract

Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.Acetylxylan esterase (AXE; EC3.1.1.72) hydrolyzes the acetyl groups from acetylated xylan [1,2]. AXE has been proposed to be the initial enzyme in the biodegradation process of cellulose acetate (CA), a plastic used for membrane materials, by decreasing the degree of substitution (DS) by acetate [3][4][5].We studied AXE from Caldanaerobacter subterraneus subsp. tengcongensis (a synonym for Thermoanaerobacter tengcongensis MB4; TTE0866) because this enzyme can uniquely deacetylate CA with a high DS of up to 2.45 [6]. TTE0866 is classified as a carbohydrate esterase family 4 (CE4) by carbohydrate-active Abbreviations AEX, anion-exchange chromatography; AXE, acetylxylan esterase; BSA, bovine serum albumin; CA, cellulose acetate; CD, circular dichroism; CE, carbohydrate esterase; DS, degree of substitution; GlcNAc, N-acetylglucosamine; HIC, hydrophobic interaction chromatography; IDR, intrinsically disordered region; IMAGEJ, image processing and analysis in java; Ni-NTA, nickel-nitrilotriacetic acid affinity chromatography; NTR, N-terminal region; PONDR, predictor of naturally disordered regions; SEC, size exclusion chromatography; WT, wild-type.