title: Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7.
abstract: A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcEP228A) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227-228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222-226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis-to-trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694-708. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, 600036, Tamil Nadu, India.
authors.forename: Mrityunjay K
authors.initials: MK
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, 600036, Tamil Nadu, India.
authors.forename: Narayanan
authors.identifier: 0000-0002-8144-1566
authors.initials: N
authors.lastname: Manoj
mesh_terms: D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000737:chemistry / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D011392:Proline; D000115:Acetylesterase; D000409:Alanine
keywords: cephalosporin C deacetylase; cis proline; cis-trans conformers; crystal structure; mutation; thermal stability