Reference "An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts"

Reference ID

54989

Title

An FMN hydrolase of the haloacid dehalogenase superfamily is active in plant chloroplasts

Source

J Biol Chem, 2011, vol. 286, pp. 42091-42098

Authors (5)

Rawat-R	Sandoval-F-J
Wei-Z	Winkler-R
Roje-S

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 approximately 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.