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.
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.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Chang Woo
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Se Jong
authors.initials: SJ
authors.lastname: Han
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Sung Gu
authors.initials: SG
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Pukyong National University, Busan 608-739, Republic of Korea.
authors.forename: Hak Jun
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology*; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002942:Circular Dichroism; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000737:chemistry*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D017931:DNA Primers; D005486:Flavin Mononucleotide; D002262:Carboxy-Lyases
keywords: 3-octaprenyl-4-hydroxybenzoate carboxy-lyase; Colwellia psychrerythraea 34H; aromatic acid decarboxylase