title: Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum.
abstract: The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis was used to generate variants with deactivated nucleophilic elbows and the functional promiscuity was analyzed. In silico analysis together with enzymological characterization interestingly showed that each nucleophilic elbow formed a local active site with varied substrate specificities and affinities. To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases.
authors.affiliation: Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway. Electronic address: gupta.udatha@outlook.com.
authors.forename: D B R K Gupta
authors.initials: DB
authors.lastname: Udatha
authors.affiliation: Center for Biological Sequence Analysis, DTU-Systems Biology, Building 208, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Karina Marie
authors.initials: KM
authors.lastname: Madsen
authors.affiliation: School of Biological Sciences, The University of Hong Kong, Kadoorie Biological Sciences Building, Hong Kong.
authors.forename: Gianni
authors.initials: G
authors.lastname: Panagiotou
authors.affiliation: Department of Chemical and Biological Engineering, Industrial Biotechnology, Chalmers University of Technology, Kemivägen 10, SE-412 96 Gothenburg, Sweden.
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Olsson
mesh_terms: D020134:Catalytic Domain / Q000235:genetics*; D016384:Consensus Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics*; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods
chemical_list: D004950:Esterases
keywords: Active sites; Esterase; Nucleophilic elbow; Protein modeling