Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity.

Abbreviations

ACP: acyl carrier protein; CAPS: N-cyclohexyl-3-aminopropanesulfonic acid; CoA: coenzyme A; CYMAL-6: 6-cyclohexyl-1-hexyl-β-D-maltoside; DDM: n-dodecyl-β-D-maltoside; DTNB: 5,5´-dithiobis(2-nitrobenzoic acid); EPA: eicosapentaenoic acid; G3P: glycerol 3-phosphate; HEPES: N-2-hydroxyethylpiperazine-N´-2-ethanesulfonic acid; LPA: lysophosphatidic acid; MS: mass spectrometry; PA: phosphatidic acid.

中文翻译：

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来自嗜热嗜热菌HB8的溶血磷脂酸酰基转移酶显示底物混杂。

溶血磷脂酸酰基转移酶是将脂肪酰基引入磷脂的sn -2位置的磷脂生物合成酶。在确定脂质膜的理化性质和调节膜蛋白功能方面，其底物选择性在生理上很重要。但是，尚不清楚这些酶如何识别各种脂肪酸。最近报道了成功的细菌溶血磷脂酸酰基转移酶（PlsCs）的纯化，为详细分析其反应机理铺平了道路。在这里，我们从嗜热菌Thermus thermophilus中纯化并鉴定了PlsCHB8。尽管支链酰基是该细菌磷脂的主要成分，但这种完整的膜蛋白即使在溶解和纯化后仍保持活性，并显示出对饱和，不饱和和甲基支链脂肪酸的反应性。多重序列比对显示该酶的N-末端比具有确定的底物选择性的PlsC的N-末端短，表明缩短的N-末端赋予了底物混杂性。

缩略语

ACP：酰基载体蛋白；CAPS：N-环己基-3-氨基丙烷磺酸；CoA：辅酶A；CYMAL-6：6-环己基-1-己基-β - D-麦芽糖苷；DDM：正十二烷基-β- D-麦芽糖苷；DTNB：5,5′-二硫代双（2-硝基苯甲酸）；EPA：二十碳五烯酸；G3P：3-磷酸甘油；HEPES：Ñ -2-羟乙基哌Ñ '-2-乙磺酸; LPA：溶血磷脂酸；MS：质谱。PA：磷脂酸。