First-time crystallization and preliminary X-ray crystallographic analysis of a bacterial-archaeal type UMP kinase, a key enzyme in microbial pyrimidine biosynthesis
✍ Scribed by Clara Marco-Marín; Juan Manuel Escamilla-Honrubia; Vicente Rubio
- Publisher
- Elsevier Science
- Year
- 2005
- Tongue
- English
- Weight
- 725 KB
- Volume
- 1747
- Category
- Article
- ISSN
- 1570-9639
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✦ Synopsis
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 UMPKencoding 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 2.4-2 resolution. Space group was I23 (a=b=c=144.95 2), 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.