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 <em>Colwellia psychrerythraea</em> 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.