Identification and biochemical characterization of a unique Mn 2+ -dependent UMP kinase from Helicobacter pylori
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ORIGINAL PAPER
Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori Mon-Juan Lee • Liu Chien-Liang • Ju-Ying Tsai • Wae-Ting Sue • Wan-Shun Hsia • Haimei Huang
Received: 7 February 2010 / Revised: 20 May 2010 / Accepted: 10 June 2010 / Published online: 3 July 2010 Ó Springer-Verlag 2010
Abstract Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg2?-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn2? over Mg2?, which may be related to a conformational difference between the Mn2?-bound and Mg2?bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg2? or Mn2?, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg2?-bound and Mn2?-bound H. pylori UMP kinase may be activated by GTP through different mechanisms. Keywords Helicobacter pylori Uridine monophosphate UMP kinase Manganese preference
Communicated by David Kelly. M.-J. Lee L. Chien-Liang J.-Y. Tsai W.-T. Sue W.-S. Hsia H. Huang (&) Institute of Biotechnology, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 30013, Taiwan e-mail: [email protected] M.-J. Lee Department of BioScience Technology, Chang Jung Christian University, Tainan, Taiwan
Introduction Uridine monophosphate (UMP) kinase represents one of the central enzymes required for pyrimidine nucleotide biosynthesis in bacteria. It has been proven to be indispensable for growth in both Gram-negative species such as Escherichia coli and Gram-positive species such as Streptococcus pneumoniae (Yamanaka et al. 1992; Jong et al. 1993; Serina et al. 1995; Fassy et al. 2004). In the presence of Mg2?, UMP kinase catalyzes the reversible transfer of the c-phosphoryl group from a nucleoside triphosphate, generally ATP, to UMP according to the scheme: UMP þ ATP Mg2þ UDPþ ADP Mg2þ . UDP is in turn converted to nucleoside triphosphate (UTP) by nucleoside diphosphate kinase (Serina et al. 1995; Gagyi et al. 2003; Fassy et al. 2004; Evrin et al. 2007). On the other hand, a bifunctional UMP/CMP kinase was commonly used in eukaryotic species to phosphorylate both UMP and CMP (Zhou and Thornburg 1998), but there was no closely related counterpart in bacteria. Prokaryotic UMP kinases are thus believed to be potential targets for antimicrobial drug development. UMP kinase from the Gram-negative E. coli is the most studied species of the UMP kinase family. It was found that E. coli UMP kinase is subjected to allosteric
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