TY - JOUR
T1 - Crystal structures of an archaeal thymidylate kinase from Sulfolobus tokodaii provide insights into the role of a conserved active site Arginine residue
AU - Biswas, Ansuman
AU - Shukla, Arpit
AU - Vijayan, R. S.K.
AU - Jeyakanthan, Jeyaraman
AU - Sekar, Kanagaraj
N1 - Publisher Copyright:
© 2016 Elsevier Inc.
PY - 2017/3/1
Y1 - 2017/3/1
N2 - 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.
AB - 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.
KW - Alanine scanning
KW - Allowed and disallowed conformation
KW - Conformational rearrangement
KW - Crystal structure
KW - Molecular dynamics
UR - http://www.scopus.com/inward/record.url?scp=85008234732&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85008234732&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2016.12.001
DO - 10.1016/j.jsb.2016.12.001
M3 - Article
C2 - 27940092
AN - SCOPUS:85008234732
SN - 1047-8477
VL - 197
SP - 236
EP - 249
JO - Journal of Structural Biology
JF - Journal of Structural Biology
IS - 3
ER -