Fah1p, a Saccharomyces cerevisiae cytochrome b(5) fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b(5) domain both function in the alpha-hydroxylation of sphingolipid-associated very long chain fatty acids
Fah1p, a Saccharomyces cerevisiae cytochrome b(5) fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b(5) domain both function in the alpha-hydroxylation of sphingolipid-associated very long chain fatty acids
复制标题
DOI：
10.1074/jbc.272.45.28281
复制
发表时间：
1997-11-07
影响因子：
4.8
通讯作者：
Martin, CE
中科院分区：
文献类型：
--
作者：
Mitchell, AG;Martin, CE
在酿酒酵母基因组数据库中搜索细胞色素B（5）样序列，发现了一个1.152-脱氢酶对开放阅读框，位于染色体XIII上的基因座YMR 272 C（FAH 1）。该基因编码384个氨基酸的蛋白质，氨基末端有一个细胞色素B（5）结构域，其B（5）核心结构域与酵母微粒体细胞色素B（5）核心结构域的同源性为52%，相似性为70%，与OLE 1的B（5）核心结构域的同源性为35%，相似性为54%。酿酒酵母Delta-9脂肪酸去饱和酶的表达;大肠杆菌中的酿酒酵母FAH 1细胞色素B（5）结构域产生一种可溶性蛋白质，其显示细胞色素B（5）的典型氧化与还原的差分吸收光谱。Fah 1 p的序列分析揭示了与Ole 1 p的其他相似性，预测两种蛋白质都具有两个疏水结构域，每个结构域能够跨膜两次，Fah 1 p与Ole 1 p的相似性表明Fah 1 p在脂肪酸的生物合成或修饰中起作用。酿酒酵母没有给出任何可见的表型，并且在最丰富的长链饱和和不饱和14-18碳脂肪酸种类的含量或分布上没有观察到差异，然而，北方印迹分析表明该基因以低得多的水平表达（类似于150倍）比OLE 1基因，这表明它可能作用于一个较小的脂肪酸子集，对鞘脂衍生的极长链脂肪酸的分析显示，在基因破坏的fah 1 Delta菌株中，α-HO 26：0减少约40倍，而26：0互补增加，S. fah 1 Delta菌株中的酿酒酵母FAH 1基因将α-HO 26：0脂肪酸恢复到野生型水平，还鉴定了该基因在其他物种中的许多同源物，不含细胞色素B（5）结构域的拟南芥FAH 1基因在fah 1 Delta菌株中的表达产生了约25倍的α-HO 26：0增加，并降低了其26-碳前体的水平，这表明它使用替代的电子转移机制在非常长链的脂肪酸羟基化中起作用。
A search of the Saccharomyces cerevisiae genome data base for cytochrome b(5)-like sequences identified a 1.152-kilobase pair open reading frame, located on chromosome XIII at locus YMR272C (FAH1). That gene encodes a putative 384-amino acid protein with an amino-terminal cytochrome b(5) domain, The b(5) core domain shows a 52% identity and 70% similarity to that of the yeast microsomal cytochrome b(5) and a 35% identity and 54% similarity to the b(5) core domain of OLE1, the S. cerevisiae Delta-9 fatty acid desaturase, Expression of the S. cerevisiae FAH1 cytochrome b(5) domain in Escherichia coli produces a soluble protein that exhibits the typical oxidized versus reduced differential absorbance spectra of cytochrome b(5).Sequence analysis of Fah1p reveals other similarities to Ole1p, Both proteins are predicted to have two hydrophobic domains, each capable of spanning the membrane twice, and both have the HX(2-3)(XH)H motifs that are characteristic of membrane-bound fatty acid desaturases, These similarities to Ole1p suggested that Fah1p played a role in the biosynthesis or modification of fatty acids.Disruption of the FAH1 gene in S. cerevisiae did not give any visible phenotype, and there was no observable difference in content or distribution of the most abundant long chain saturated and unsaturated 14-18-carbon fatty acid species, Northern blot analysis, however, showed that this gene is expressed at much lower levels (similar to 150-fold) than the OLE1 gene, suggesting that it might act on a smaller subset of fatty acids, Analysis of sphingolipid-derived very long chain fatty acids revealed an approximately 40 fold reduction of alpha-HO 26:0 and a complementary increase in 26:0 in the gene-disrupted fah1 Delta strain, GAL1 expression of the S. cerevisiae FAH1 genes in the fah1 Delta strain restores alpha-HO 26:0 fatty acids to wild type levels, Also identified are a number of homologs to this gene in other Species, Expression of an Arabidopsis thaliana FAH1 gene, which does not contain the cytochrome b(5) domain, in the fah1 Delta strain produced an approximately 25-fold increase in alpha-HO 26:0 and reduced the levels of its 26-carbon precursor, suggesting that it functions in very long chain fatty acid hydroxylation using an alternate electron transfer mechanism.