A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan

• Published in: The Journal of biological chemistry Vol. 294; no. 46; pp. 17339 - 17353
• Main Authors: Holck, Jesper, Fredslund, Folmer, Møller, Marie S., Brask, Jesper, Krogh, Kristian B.R.M., Lange, Lene, Welner, Ditte H., Svensson, Birte, Meyer, Anne S., Wilkens, Casper
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-11-2019; American Society for Biochemistry and Molecular Biology
• Subjects: Arabinose - chemistry; arabinoxylan; carbohydrate esterase family 1; carbohydrate-binding module; Carboxylesterase - chemistry; Carboxylesterase - genetics; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - ultrastructure; Coumaric Acids - chemistry; crystal structure; Crystallography, X-Ray; enzyme catalysis; enzyme mechanism; Enzymology; Escherichia coli - chemistry; Escherichia coli - enzymology; ferulic acid esterase; Hydrolysis; molecular docking; molecular dynamics; Oligosaccharides - chemistry; Polysaccharides - chemistry; Protein Conformation; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - ultrastructure; structure–function; Substrate Specificity; Surface Plasmon Resonance; Waste Water - chemistry; Xylans - chemistry; ferulic acid esterase; molecular docking; arabinoxylan; carbohydrate-binding module; crystal structure; carbohydrate esterase family 1; enzyme catalysis; enzyme mechanism; molecular dynamics; structure–function
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009523

Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 μm and that it does not bind to starch mimics, β-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a β-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 Å resolution) and wtsFae1B (1.98 Å) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1–CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.