Journal article
An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts
The Journal of biological chemistry, Vol.286(49), pp.42091-42098
12/09/2011
Handle:
https://hdl.handle.net/2376/109437
PMCID: PMC3234908
PMID: 22002057
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 Mg2+-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.
Background: Flavin nucleotide metabolism in plants is incompletely understood.
Results: Gene At1g79790 encodes an FMN-specific hydrolase (AtcpFHy1) that localizes to plastids.
Conclusion: This is the first study reporting cloning and characterization of an FMN hydrolase from plastids.
Significance: This study advances the knowledge of flavin nucleotide metabolism in plants.
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Details
- Title
- An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts
- Creators
- Renu Rawat - Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164Francisco J Sandoval - Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164Zhaoyang Wei - Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164Robert Winkler - Departamento de Biotecnología y Tecnología de Alimentos, Instituto Tecnológico y de Estudios Superiores de Monterrey, 64849 Monterrey, Nuevo León, MexicoSanja Roje - Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164
- Publication Details
- The Journal of biological chemistry, Vol.286(49), pp.42091-42098
- Academic Unit
- Biological Chemistry, Institute of
- Publisher
- Elsevier Inc
- Identifiers
- 99900547050401842
- Language
- English
- Resource Type
- Journal article