The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1∼6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension (LNTE, residues 26-283) between a putative signal peptide (residues 1-25) and a C-terminal esterase domain (residues 284-510). A putative esterase from Candidatus Solibacter usitatus (CSu-Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC-Est1. To examine whether LC-Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC-Est1 (residues 26-510), LC-Est1C (residues 284-510), and LC-Est1C* (residues 304-510) were overproduced in E. coli, purified, and characterized. LC-Est1C* was only used for structural analysis. The crystal structure of LC-Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC-Est1C was lower than that of LC-Est1 by 60%, although its substrate specificity was similar to that of LC-Est1. LC-Est1C was less stable than LC-Est1 by 3.3°C. These results suggest that LNTE of LC-Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.
Keywords: N-terminal extension; crystal structure; esterase; metagenome; stability.
© 2014 The Protein Society.