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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
Other Titles
호냉성 박테리아인 Colwellia psychrerythraea 34H 유래 aromatic acid decarboxylase (CpsUbiX) 단백질의 정제, 결정화 및 X-선 결정학적 연구
Authors
Lee, Jun Hyuck
Kim, Hak Jun
Do, Hackwon
Lee, Chang Woo
Han, Se Jong
Lee, Sung Gu
Park, Hyun
Keywords
3-octaprenyl-4-hydroxybenzoate carboxy-lyase; Aromatic acid decarboxylase; Colwellia psychrerythraea 34H; X-ray crystallography
Issue Date
2014
Citation
Lee, Jun Hyuck, 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 Section F: Structural Biology Communications, 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.
URI
http://repository.kopri.re.kr/handle/201206/7251
DOI
http://dx.doi.org/10.1107/S2053230X1303447X
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