First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis.
UMP phosphorylation, a key step for pyrimidine nucleotide biosynthesis, is catalyzed in bacteria by UMP kinase (UMPK), an enzyme specific for UMP that is dissimilar to the eukaryotic UMP/CMP kinase or to other nucleoside monophosphate kinases. UMPK is allosterically regulated and participates in pyrimidine-triggered gene repression. As first step towards determining UMPK structure, the putative UMPK-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus was cloned and overexpressed in Escherichia coli. The protein product was purified and confirmed to be a genuine UMPK. It was crystallized at 294 K in hanging drops by the vapor diffusion technique using 3.5-4 M Na formate. Cubic 0.2-mm crystals diffracted synchrotron X-rays to 2.4-angstroms resolution. Space group was I23 (a=b=c=144.95 angstroms), and the asymmetric unit contained two monomers, with 52% solvent content. The self-rotation function suggests that the enzyme is hexameric, which agrees with biochemical studies on bacterial UMPKs.
Marco-Marín
Clara
C
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas (IBV-CSIC), C/Jaime Roig 11, 46010-Valencia, Spain.
Escamilla-Honrubia
Juan Manuel
JM
Rubio
Vicente
V
D002384:Catalysis; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D004058:Diffusion; D004926:Escherichia coli / Q000235:genetics; D005057:Eukaryotic Cells / Q000378:metabolism; D020745:Genome, Archaeal; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011743:Pyrimidines / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012997:Solvents; D013696:Temperature
D011743:Pyrimidines; D011994:Recombinant Proteins; D012997:Solvents; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; C030986:pyrimidine