Successful data recovery from oscillation photographs containing strong polycrystalline diffraction rings from an unknown small-molecule contaminant: preliminary structure solution of Salmonella typhimurium pyridoxal kinase (PdxK).
Pyridoxal kinase (PdxK; EC 2.7.1.35) belongs to the phosphotransferase family of enzymes and catalyzes the conversion of the three active forms of vitamin B6, pyridoxine, pyridoxal and pyridoxamine, to their phosphorylated forms and thereby plays a key role in pyridoxal 5'-phosphate salvage. In the present study, pyridoxal kinase from Salmonella typhimurium was cloned and overexpressed in Escherichia coli, purified using Ni-NTA affinity chromatography and crystallized. X-ray diffraction data were collected to 2.6 Å resolution at 100 K. The crystal belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 65.11, b = 72.89, c = 107.52 Å. The data quality obtained by routine processing was poor owing to the presence of strong diffraction rings caused by a polycrystalline material of an unknown small molecule in all oscillation images. Excluding the reflections close to powder/polycrystalline rings provided data of sufficient quality for structure determination. A preliminary structure solution has been obtained by molecular replacement with the Phaser program in the CCP4 suite using E. coli pyridoxal kinase (PDB entry 2ddm) as the phasing model. Further refinement and analysis of the structure are likely to provide valuable insights into catalysis by pyridoxal kinases.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
G
G
Deka
Biochemistry Department, Indian Institute of Science, Bangalore 560 012, India.
J N
JN
Kalyani
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
J F
JF
Benazir
Biochemistry Department, Indian Institute of Science, Bangalore 560 012, India.
H S
HS
Savithri
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India.
M R N
MR
Murthy
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D003625:Data Collection / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011487:Protein Conformation; D011731:Pyridoxal Kinase / Q000737:chemistry* / Q000302:isolation & purification*; D012486:Salmonella typhimurium / Q000201:enzymology*
D004364:Pharmaceutical Preparations; D011731:Pyridoxal Kinase
Salmonella typhimurium; pyridoxal kinase