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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

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Title
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
Authors
Do, Hackwon
Lee, Chang Woo
Han, Se Jong
Lee, Sung Gu
Kim, Hak Jun
Park, Hyun
Lee, Jun Hyuck
Subject
Biochemistry & Molecular Biology; Biophysics; Crystallography
Keywords
3-octaprenyl-4-hydroxybenzoate carboxy-lyas; Aromatic acid decarboxylase; Colwellia psychrerythraea 34H; X-ray crystallography
Issue Date
2014
Citation
Do, Hackwon., et al. 2014. 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. Acta Crystallographica, F(70): 215?220.
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, we found that the V47S single mutation resulted in a loss of FMN binding, resulting in the production of the FMN-free CpsUbiX protein. This mutation likely destabilizes FMN-protein interactions without affecting overall structural folding. To fully characterize the conformational changes upon FMN binding and 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 vapor diffusion method. Furthermore, complete diffraction data sets of FMN-bound (space group C2221) and FMN-free (space group P23) forms were obtained to 2.0 and 1.76 A resolution, respectively.
DOI
10.1107/S2053230X1303447X
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