Cloning and characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide
Cloning and characterization of a Saccharomyces cerevisiae alkaline ceramidase with specificity for dihydroceramide
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DOI：
10.1074/jbc.m003683200
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发表时间：
2000-10-06
影响因子：
4.8
通讯作者：
Obeid, LM
中科院分区：
文献类型：
--
作者：
Mao, CG;Xu, RJ;Obeid, LM
在先前的研究中，我们报道了酿酒酵母基因YPC 1编码具有双重活性的碱性神经酰胺酶，催化酵母神经酰胺的水解和合成（Mao，C.，徐，R.，Bielawska，A.，和奥贝德，L.，M.，（2000）J.Biol.Chem.275，6876-6884）。在这项研究中，我们已经确定了一个YPC 1同源的S。我们发现这两种神经酰胺酶具有不同的底物特异性，例如YPC 1 p优先水解植物神经酰胺，而新的神经酰胺酶YDC 1 p优先水解二氢神经酰胺，而植物神经酰胺仅轻微水解。两种酶都不水解不饱和的神经酰胺。与YPC 1 p相反，YDC 1 p在体外仅具有催化由游离脂肪酸和二氢鞘氨醇形成二氢神经酰胺的微小逆转活性，并且与植物鞘氨醇没有活性。YDC 1 p的过表达在非应激酵母细胞中没有逆转活性，但是像YPC 1 p一样抑制伏马菌素B1对生长的抑制，尽管更温和。YDC 1和YPC 1的缺失或两者的缺失对生长没有明显影响，表明这两个基因都不是必需的，然而，Δ YPC 1缺失突变体而不是Δ YPC 1缺失突变体对热胁迫敏感，表明二氢神经酰胺而不是植物神经酰胺在热胁迫响应中的作用，并表明这两种酶具有不同的生理功能。
In a previous study, we reported that the Saccharomyces cerevisiae gene YPC1 encodes an alkaline ceramidase with a dual activity, catalyzing both hydrolysis and synthesis of yeast ceramide (Mao, C., Xu, R., Bielawska, A., and Obeid, L., M., (2000) J. Biol. Chem. 275, 6876-6884). In this study, we have identified a YPC1 homologue in S. cerevisiae that also encodes an alkaline ceramidase, We show that these two ceramidases have different substrate specificity, such that YPC1p preferentially hydrolyzes phytoceramide, whereas the new ceramidase YDC1p hydrolyzes dihydroceramide preferentially and phytoceramide only slightly. Neither enzyme hydrolyzes unsaturated mammalian-type ceramide. In contrast to YPC1p, YDC1p had only minor in vitro reverse activity of catalyzing dihydroceramide formation from a free fatty acid and dihydrosphingosine and no activity with phytosphingosine, Overexpression of YDC1p had no reverse activity in non-stressed yeast cells, but like YPC1p suppressed the inhibition of growth by fumonisin B1 albeit more modestly. Deletion of YDC1 and YPC1 or both did not apparently affect growth, suggesting neither gene is essential, However, the Delta ydc1 deletion mutant but not the Delta ypc1 deletion mutant was sensitive to heat stress, indicating a role for dihydroceramide but not phytoceramide in heat stress responses, and suggesting that the two enzymes have distinct physiological functions.