STEAROYL-ACYL CARRIER PROTEIN DELTA-9 DESATURASE FROM RICINUS-COMMUNIS IS A DIIRON-OXO PROTEIN
STEAROYL-ACYL CARRIER PROTEIN DELTA-9 DESATURASE FROM RICINUS-COMMUNIS IS A DIIRON-OXO PROTEIN
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DOI：
10.1073/pnas.90.6.2486
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发表时间：
1993-03-15
影响因子：
11.1
通讯作者：
MUNCK, E
中科院分区：
文献类型：
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作者：
FOX, BG;SHANKLIN, J;MUNCK, E
在大肠杆菌中表达编码来自蓖麻的硬脂酰-酰基载体蛋白DELTA 9去饱和酶（EC 1.14.99.6）的基因。纯化的催化活性酶含有四个原子的铁每同型二聚体。用穆斯堡尔谱学研究了去饱和酶的两种氧化态，外加磁场高达6.0T。这些研究结果表明，氧化酶含有两个（相同的）集群组成的一对反铁磁耦合（J > 60 cm-1，H = JS 1.S2）Fe 3+网站。二铁簇表现出从300至355 nm的吸收带;另外的叠氮化物引起的电荷转移带在450 nm处。在连二亚硫酸盐存在下，团簇被还原为二亚铁态。添加硬脂酰辅酶A和O2使簇返回二铁状态。这些属性是一致的分配去饱和酶的类O2-活化蛋白质含有二铁氧簇，最显着的核糖核苷酸还原酶和甲烷单加氧酶羟化酶。这三种催化不同的蛋白质的一级结构的比较揭示了一个保守的氨基酸序列对-（Asp/Glu）-Glu-Xaa-Arg-His-分开的几乎等于100个氨基酸。由于这些蛋白质中的每一个都可以催化未活化的C-H键的O2依赖性裂解，我们建议这些氨基酸序列代表用于创建反应性催化中间体的生物基序。因此，真核脂肪酸去饱和可以通过酶促生成衍生自二铁簇的高价铁-氧代物质来进行。
A gene encoding stearoyl-acyl carrier protein DELTA9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mossbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by almost-equal-to 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C-H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.