Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes
Identification of the gene encoding alkylglycerol monooxygenase defines a third class of tetrahydrobiopterin-dependent enzymes
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DOI：
10.1073/pnas.1002404107
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发表时间：
2010-08-03
影响因子：
11.1
通讯作者：
Werner, Ernst R.
中科院分区：
文献类型：
--
作者：
Watschinger, Katrin;Keller, Markus A.;Werner, Ernst R.
烷基甘油单加氧酶（甘油基-醚单加氧酶，EC 1.14.16.5）是唯一已知的切割醚脂质的O-烷基键的酶，醚脂质是脑膜的基本组分，保护眼睛免受白内障，干扰或介导信号传导过程，并且是精子发生所需的。与苯丙氨酸羟化酶、酪氨酸羟化酶、色氨酸羟化酶和一氧化氮合酶沿着，烷基甘油单加氧酶是依赖于四氢生物蝶呤的五种已知酶促反应之一。虽然在1964年首次描述，但迄今为止还没有为这种酶分配序列，因为它在蛋白质纯化尝试时失去了活性。使用转染至CHO细胞的大鼠肝脏表达文库的质粒池进行的功能性文库筛选也不成功。因此，我们选择了人类候选基因的生物信息学方法和蛋白质组学分析的部分纯化的酶和测试烷基甘油单加氧酶的活性在CHO细胞转染表达质粒。跨膜蛋白195是一种在动物体内存在的功能未知的膜蛋白，编码四氢生物蝶呤依赖的烷基甘油单加氧酶。通过将跨膜蛋白195 cRNA注射到非洲爪蟾卵母细胞中证实了该序列分配。跨膜蛋白195与芳香族氨基酸羟化酶或一氧化氮合酶没有序列同源性，但含有脂肪酸羟化酶基序。该基序存在于含有二铁中心的酶中，并且在脂肪族碳原子处进行脂质的羟基化，如烷基甘油单加氧酶。这一序列分配表明，烷基甘油单加氧酶形成一个独特的第三组之间的四氢生物蝶呤依赖性酶。
Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes.