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J. Biol. Chem., Vol. 280, Issue 46, 38337-38345, November 18, 2005

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An FMN Hydrolase Is Fused to a Riboflavin Kinase Homolog in Plants^*

From the Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164

Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg²⁺ in the assays, as expected for a member of the Mg²⁺-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties.

Received for publication, January 11, 2005 , and in revised form, September 19, 2005.

^* This work was supported by a start-up fund from Washington State University (to S. R.). 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBIData Bank with accession number(s) AY878327 [GenBank] (A. thaliana FMN/FHy).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Inst. of Biological Chemistry, Washington State University, Pullman, WA 99164. Tel.: 509-335-3008; Fax: 509-335-7643; E-mail: sanja{at}wsu.edu.

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