Abstract：
Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA), an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid-binding domain of CFAS enzymes, perhaps suggesting distinct membrane-binding properties among different orthologs. This work lays an important foundation for further characterization of CFAS in P. aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.
摘要：
环丙烷脂肪酸合酶(CFAS)催化细菌膜内不饱和脂肪酸转化为环丙烷脂肪酸(CFA)。这种修饰改变了膜的生物物理特性，并且与几种人类病原体的毒力相关。尽管CFAS酶在调节细菌应激反应中起着核心作用，CFAS酶家族的机制特性和CFA生物合成的后果在大多数细菌中仍未表征。我们报告了铜绿假单胞菌（PA）的CFAS酶的第一个特征，PA是一种机会性人类病原体，具有复杂的膜生物学特性，通常与抗菌素耐药性和对各种外部应激源的高耐受性有关。我们证明了CFA是由PA中的单个酶产生的，并且cfas基因表达在向稳定期过渡和对氧化应激的反应中上调。PA脂质提取物的分析显示，随着PA细胞进入静止期，CFA产量大幅增加，并有助于确定体外测定的最佳膜组成。纯化的PA-CFAS酶形成稳定的同二聚体，并优先修饰磷脂酰甘油脂质底物和具有较高含量不饱和酰基链的膜。跨细菌门的生物信息学分析显示CFAS酶的脂质结合域内高度不同的氨基酸序列，也许表明不同直系同源物之间具有不同的膜结合特性。本工作为进一步鉴定铜绿假单胞菌中的CFAS和研究不同细菌的CFAS酶的功能差异奠定了重要的基础。
