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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.

SUBMITTER: Rawat R

PROVIDER: S-EPMC3234908 | biostudies-other | 2011 Dec

REPOSITORIES: biostudies-other

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

Rawat Renu R Sandoval Francisco J FJ Wei Zhaoyang Z Winkler Robert R Roje Sanja S

The Journal of biological chemistry 20111014 49

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 chr ...[more]

PMID: 22002057

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Project description:The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k(cat)/K(m) value for pyrophosphate of ~1 × 10(5) M(-1) s(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure-guided site-directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue, ?-phosphoglucomutase, control the leaving group size (phosphate vs glucose phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases.

Project description:Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C-P bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 A X-ray crystal structure of the mutant Lys53Arg complexed with Mg2+ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain-core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH4 in the presence of Pald was determined at 2.4A resolution to reveal N epsilon-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C2 of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain N epsilon-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P-C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P-C bond cleavage.

Dataset Information

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

Publications

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

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