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J. Biol. Chem., Vol. 280, Issue 7, 5274-5280, February 18, 2005

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A Nudix Enzyme Removes Pyrophosphate from Dihydroneopterin Triphosphate in the Folate Synthesis Pathway of Bacteria and Plants^*

Sebastian M. J. Klaus, Arno Wegkamp¶, Wilbert Sybesma¶, Jeroen Hugenholtz¶, Jesse F. Gregory, III||, and Andrew D. Hanson**

From the Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, the ^¶Department of Flavor, Nutrition and Natural Ingredients, Wageningen Centre for Food Sciences, NIZO Food Research, Kernhemseweg 2, P. O. Box 20, 6710, BA Ede, The Netherlands, and the ^||Department of Food Science and Human Nutrition, University of Florida, Gainesville, Florida 32611

Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a K_m value of 2 µM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (1 mM) of Mg²⁺, and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg²⁺. Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg²⁺-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn²⁺ (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482–50486). However, we found that this activity is negligible at physiological levels of Mn²⁺ and that, with 1 mM Mg²⁺, AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates.

Received for publication, December 7, 2004

^* This work was supported in part by the Florida Agricultural Experimental Station, by an endowment from the C. V. Griffin, Sr., Foundation, and by Grant 2005-35318-15228 from the National Research Initiative Competitive Grants Program of the United States Department of Agriculture. This work was approved for publication as Journal Series number R-10630. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table I.

Both authors contributed equally to this work.

^** To whom correspondence should be addressed: Horticultural Sciences Dept., University of Florida, P. O. Box 110690, Gainesville, FL 32611. Tel.: 352-392-1928; Fax: 352-392-5653; E-mail: adha{at}mail.ifas.ufl.edu.

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