title: Crystal structures of an archaeal thymidylate kinase from Sulfolobus tokodaii provide insights into the role of a conserved active site Arginine residue.
abstract: Thymidylate kinase (TMK) is a key enzyme that plays an important role in DNA synthesis. Therefore, it serves as an attractive therapeutic target for the development of antibacterial, antiparasitic and anticancer drugs. Herein, we report the biochemical characterization and crystal structure determination of thymidylate kinase from a hyperthermophilic organism Sulfolobus tokodaii (StTMK) in its apo and ADP-bound forms. Our study describes the first three-dimensional structure of an archaeal TMK. StTMK is a thermostable enzyme with optimum activity at 80°C. Despite the overall similarity to homologous TMKs, StTMK structures revealed several residue substitutions at the active site. However, enzyme assays demonstrated specificity to its natural substrates ATP and dTMP. Analysis of the structures also revealed multiple conformational states of Arg93 which is located at the reaction centre and is a part of the highly conserved DRX motif. Only one of these states was found to be suitable for the proper positioning of the α-phosphate group of dTMP at the active site. Computational alanine scanning and MM/PBSA binding energy calculation revealed the importance of Arg93 side chain in substrate binding. Subsequent site directed mutagenesis at this position to an Ala resulted in the loss of activity. Thus, the computational and biochemical studies reveal the importance of Arg93 for enzyme function, while the different conformational states of Arg93 observed in the structural studies imply its regulatory role in the catalytically competent placement of dTMP.
authors.affiliation: Department of Physics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Ansuman
authors.initials: A
authors.lastname: Biswas
authors.affiliation: Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O., Bangalore 560064, India.
authors.forename: Arpit
authors.initials: A
authors.lastname: Shukla
authors.affiliation: Departement of Chemistry, College of Science and Technology, Temple University, 1925 N. 12th Street, Philadelphia, PA 19122-1801, USA.
authors.forename: R S K
authors.initials: RSK
authors.lastname: Vijayan
authors.affiliation: Department of Bioinformatics, Alagappa University, Karaikudi 630003, India.
authors.forename: Jeyaraman
authors.initials: J
authors.lastname: Jeyakanthan
authors.affiliation: Department of Computational and Data Sciences, Indian Institute of Science, Bangalore 560012, India. Electronic address: sekar@cds.iisc.ac.in.
authors.forename: Kanagaraj
authors.initials: K
authors.lastname: Sekar
mesh_terms: D001105:Archaea / Q000201:enzymology*; D001120:Arginine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology; D056004:Molecular Dynamics Simulation; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
chemical_list: D001120:Arginine; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase
keywords: Alanine scanning; Allowed and disallowed conformation; Conformational rearrangement; Crystal structure; Molecular dynamics