Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis.
Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B(6) precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5'-phosphates and play an important role in the vitamin B(6) salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ;HMPP kinase-like' pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg(2+). X-ray diffraction data have been collected from crystals to 2.8 A resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P4(1)2(1)2 or P4(3)2(1)2. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 A, alpha = beta = gamma = 90 degrees ) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.
Newman
Joseph A
JA
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, England.
Das
Sanjan K
SK
Sedelnikova
Svetlana E
SE
Rice
David W
DW
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D011731:Pyridoxal Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D001426:Bacterial Proteins; D000244:Adenosine Diphosphate; D011731:Pyridoxal Kinase; D008274:Magnesium