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Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacterium Colwellia psychrerythraea 34H.

ABSTRACT: The ubiX gene (UniProtKB code Q489U8) of Colwellia psychrerythraea strain 34H has been annotated as a putative flavin mononucleotide (FMN)-dependent aromatic acid decarboxylase. Based on previous studies of homologous proteins, CpsUbiX is thought to catalyze the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to produce 2-polyprenylphenol in the ubiquinone-biosynthesis pathway using a noncovalently bound FMN molecule as a cofactor. However, the detailed mechanisms of this important enzyme are not yet clear and need to be further elucidated. In this study, it was found that the V47S single mutation resulted in a loss of FMN binding, resulting in the production of FMN-free CpsUbiX protein. This mutation is likely to destabilize FMN-protein interactions without affecting the overall structural folding. To fully characterize the conformational changes upon FMN binding and the enzymatic mechanism at the molecular level, the wild-type (FMN-bound) and V47S mutant (FMN-free) CpsUbiX proteins were purified and crystallized using the sitting-drop vapour-diffusion method. Furthermore, complete diffraction data sets for FMN-bound (space group C222(1)) and FMN-free (space group P23) forms were obtained to 2.0 and 1.76 Å resolution, respectively.

SUBMITTER: Do H

PROVIDER: S-EPMC3936454 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacterium Colwellia psychrerythraea 34H.

Do Hackwon H Lee Chang Woo CW Han Se Jong SJ Lee Sung Gu SG Kim Hak Jun HJ Park Hyun H Lee Jun Hyuck JH

Acta crystallographica. Section F, Structural biology communications 20140121 Pt 2

The ubiX gene (UniProtKB code Q489U8) of Colwellia psychrerythraea strain 34H has been annotated as a putative flavin mononucleotide (FMN)-dependent aromatic acid decarboxylase. Based on previous studies of homologous proteins, CpsUbiX is thought to catalyze the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to produce 2-polyprenylphenol in the ubiquinone-biosynthesis pathway using a noncovalently bound FMN molecule as a cofactor. However, the detailed mechanisms of this important enzyme are ...[more]

PMID: 24637760

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Project description:Isoaspartyl dipeptidase (IadA) is an enzyme that catalyzes the hydrolysis of an isoaspartyl dipeptide-like moiety, which can be inappropriately formed in proteins, between the β-carboxyl group side chain of Asp and the amino group of the following amino acid. Here, we have determined the structures of an isoaspartyl dipeptidase (CpsIadA) from Colwellia psychrerythraea, both ligand-free and that complexed with β-isoaspartyl lysine, at 1.85-Å and 2.33-Å resolution, respectively. In both structures, CpsIadA formed an octamer with two Zn ions in the active site. A structural comparison with Escherichia coli isoaspartyl dipeptidase (EcoIadA) revealed a major difference in the structure of the active site. For metal ion coordination, CpsIadA has a Glu166 residue in the active site, whereas EcoIadA has a post-translationally carbamylated-lysine 162 residue. Site-directed mutagenesis studies confirmed that the Glu166 residue is critical for CpsIadA enzymatic activity. This residue substitution from lysine to glutamate induces the protrusion of the β12-α8 loop into the active site to compensate for the loss of length of the side chain. In addition, the α3-β9 loop of CpsIadA adopts a different conformation compared to EcoIadA, which induces a change in the structure of the substrate-binding pocket. Despite CpsIadA having a different active-site residue composition and substrate-binding pocket, there is only a slight difference in CpsIadA substrate specificity compared with EcoIadA. Comparative sequence analysis classified IadA-containing bacteria and archaea into two groups based on the active-site residue composition, with Type I IadAs having a glutamate residue and Type II IadAs having a carbamylated-lysine residue. CpsIadA has maximal activity at pH 8-8.5 and 45°C, and was completely inactivated at 60°C. Despite being isolated from a psychrophilic bacteria, CpsIadA is thermostable probably owing to its octameric structure. This is the first conclusive description of the structure and properties of a Type I IadA.

Dataset Information

Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacterium Colwellia psychrerythraea 34H.

Publications

Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacterium Colwellia psychrerythraea 34H.

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