title: An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts.
abstract: FMN hydrolases catalyze dephosphorylation of FMN to riboflavin. Although these enzymes have been described in many organisms, few had their corresponding genes cloned and their recombinant proteins biochemically characterized, and none had their physiological roles determined. We found previously that FMN hydrolase activity in pea chloroplasts is Mg(2+)-dependent, suggesting an enzyme of the haloacid dehalogenase (HAD) superfamily. In this study, a new FMN hydrolase was purified by multistep chromatography after ammonium sulfate precipitation. The molecular weight of the native protein was estimated at ∼59,400, a dimer of about twice the predicted molecular weight of most HAD superfamily phosphatases. After SDS-PAGE of the partially purified material, two separate protein bands within 25-30 kDa were extracted from the gel and analyzed by nanoLC-MS/MS. Peptide sequence matching to the protein samples suggested the presence of three HAD-like hydrolases. cDNAs for sequence homologs from Arabidopsis thaliana of these proteins were expressed in Escherichia coli. Activity screening of the encoded proteins showed that the At1g79790 gene encodes an FMN hydrolase (AtcpFHy1). Plastid localization of AtcpFHy1 was confirmed using fluorescence microscopy of A. thaliana protoplasts transiently expressing the N-terminal fusion of AtcpFHy1 to enhanced green fluorescent protein. Phosphatase activity of AtcpFHy1 is FMN-specific, as assayed with 19 potential substrates. Kinetic parameters and pH and temperature optima for AtcpFHy1 were determined. A phylogenetic analysis of putative phosphatases of the HAD superfamily suggested distinct evolutionary origins for the plastid AtcpFHy1 and the cytosolic FMN hydrolase characterized previously.
authors: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164.
authors: Renu
authors: R
authors: Rawat
authors: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164.
authors: Francisco J
authors: FJ
authors: Sandoval
authors: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164.
authors: Zhaoyang
authors: Z
authors: Wei
authors: Departamento de Biotecnología y Tecnología de Alimentos, Instituto Tecnológico y de Estudios Superiores de Monterrey, 64849 Monterrey, Nuevo León, Mexico.
authors: Robert
authors: R
authors: Winkler
authors: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164. Electronic address: sanja@wsu.edu.
authors: Sanja
authors: S
authors: Roje
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D038181:FMN Reductase / Q000502:physiology*; D017343:Genes, Plant; D006867:Hydrolases / Q000737:chemistry*; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D008957:Models, Genetic; D008970:Molecular Weight; D018532:Pisum sativum / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010944:Plants / Q000378:metabolism*; D018087:Plastids / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D013696:Temperature
chemical_list: D010455:Peptides; D038181:FMN Reductase; D006867:Hydrolases; C034507:2-haloacid dehalogenase; D008274:Magnesium