From Protease to Decarboxylase THE MOLECULAR METAMORPHOSIS OF PHOSPHATIDYLSERINE DECARBOXYLASE
From Protease to Decarboxylase THE MOLECULAR METAMORPHOSIS OF PHOSPHATIDYLSERINE DECARBOXYLASE
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DOI：
10.1074/jbc.m115.642413
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发表时间：
2015-04-24
影响因子：
4.8
通讯作者：
Voelker, Dennis R.
中科院分区：
文献类型：
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作者：
Choi, Jae-Yeon;Duraisingh, Manoj T.;Voelker, Dennis R.
磷脂酰丝氨酸脱羧酶（psd）在许多原核生物和真核生物合成磷脂酰乙醇胺的过程中起着重要作用。psd是一种罕见的脱羧酶，在活性位点含有丙酮酰假基。当PSD前酶经历蛋白内水解裂解成一个大的β亚基和一个较小的α亚基时，共价附着的丙酮基片段在协同反应中形成，α亚基在其N端含有假基。PSD前酶裂解的机制一直不清楚。利用体外转录/翻译系统与可溶性诺氏疟原虫酶（PkPSD）的耦合，我们证明了丝氨酸蛋白酶抑制剂苯基甲基磺酰氟可以抑制翻译后加工。通过对多门PSD序列的比较，发现在FFXRX6RX12PXD基序中有一个独特的保守的天冬氨酸，在PXXYHXXHXP基序中有两个独特的保守的组氨酸残基，在GS（S/T）基序中有一个独特的保守的丝氨酸残基，表明PSD属于D-H-S丝氨酸蛋白酶家族。利用PkPSD的定点诱变技术探测了保守的D-H-S残基的功能。这些诱变实验的结果表明，Asp-139、His-198和Ser-308都是发生在顺式中PkPSD的内源性蛋白水解过程所必需的。此外，在所有psd中发现的GS（S/T）基序中，Gly-307残基也是必需的，而Ser/Thr-309残基则不是必需的。这些结果确定了PSD作为蛋白酶开始其生化存在的机制，这些蛋白酶执行一次自身内源性蛋白水解裂解反应，产生含有丙酮酰假基的成熟PSD。
Phosphatidylserine decarboxylase (PSDs) play a central role in the synthesis of phosphatidylethanolamine in numerous species of prokaryotes and eukaryotes. PSDs are unusual decarboxylase containing a pyruvoyl prosthetic group within the active site. The covalently attached pyruvoyl moiety is formed in a concerted reaction when the PSD proenzyme undergoes an endoproteolytic cleavage into a large beta-subunit, and a smaller alpha-subunit, which harbors the prosthetic group at its N terminus. The mechanism of PSD proenzyme cleavage has long been unclear. Using a coupled in vitro transcription/translation system with the soluble Plasmodium knowlesi enzyme (PkPSD), we demonstrate that the post-translational processing is inhibited by the serine protease inhibitor, phenylmethylsulfonyl fluoride. Comparison of PSD sequences across multiple phyla reveals a uniquely conserved aspartic acid within an FFXRX6RX12PXD motif, two uniquely conserved histidine residues within a PXXYHXXHXP motif, and a uniquely conserved serine residue within a GS(S/T) motif, suggesting that PSDs belong to the D-H-S serine protease family. The function of the conserved D-H-S residues was probed using site-directed mutagenesis of PkPSD. The results from these mutagenesis experiments reveal that Asp-139, His-198, and Ser-308 are all essential for endoproteolytic processing of PkPSD, which occurs in cis. In addition, within the GS(S/T) motif found in all PSDs, the Gly-307 residue is also essential, but the Ser/Thr-309 is non-essential. These results define the mechanism whereby PSDs begin their biochemical existence as proteases that execute one autoendoproteolytic cleavage reaction to give rise to a mature PSD harboring a pyruvoyl prosthetic group.