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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
Author(s) -
Do Hackwon,
Lee Chang Woo,
Han Se Jong,
Lee Sung Gu,
Kim Hak Jun,
Park Hyun,
Lee Jun Hyuck
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x1303447x
Subject(s) - flavin mononucleotide , chemistry , stereochemistry , cofactor , decarboxylation , biochemistry , enzyme , catalysis
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 C 222 1 ) and FMN‐free (space group P 23) forms were obtained to 2.0 and 1.76 Å resolution, respectively.