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. 2016 Jun;194(3):434-45.
doi: 10.1016/j.jsb.2016.04.008. Epub 2016 Apr 13.

An Extended Loop in CE7 Carbohydrate Esterase Family Is Dispensable for Oligomerization but Required for Activity and Thermostability

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An Extended Loop in CE7 Carbohydrate Esterase Family Is Dispensable for Oligomerization but Required for Activity and Thermostability

Mrityunjay K Singh et al. J Struct Biol. .

Abstract

The carbohydrate esterase family 7 (CE7) belonging to the α/β hydrolase superfamily contains a structurally conserved loop extension element relative to the canonical α/β hydrolase fold. This element called the β-interface loop contributes 20-30% of the total buried surface area at intersubunit interfaces of the functional hexameric state. To test whether this loop is an enabling region for the structure and function of the oligomeric assembly, we designed a truncation variant of the thermostable CE7 acetyl esterase from Thermotoga maritima (TmAcE). Although deletion of 26 out of 40 residues in the loop had little impact on the hexamer formation, the variant exhibited altered dynamics of the oligomeric assembly and a loss of thermal stability. Furthermore, the mutant lacked catalytic activity. Crystal structures of the variant and a new crystal form of the wild type protein determined at 2.75Å and 1.76Å, respectively, provide a rationale for the properties of the variant. The hexameric assembly in the variant is identical to that of the wild type and differed only in the lack of buried surface area interactions at the original intersubunit interfaces. This is accompanied by disorder in an extended region of the truncated loop that consequently induces disorder in the neighboring oxyanion hole loop. Overall, the results suggest that the β-interface loop in CE7 enzymes is dispensable for the oligomeric assembly. Rather, the loop extension event was evolutionarily selected to regulate activity, conformational flexibility and thermal stability.

Keywords: Carbohydrate esterase family 7; Crystal structure; Intersubunit interface; Loop truncation; Thermotoga maritima acetyl esterase.

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