In previous work, we identified a triple mutant of the castor (Ricinus communis) stearoyl-Acyl Carrier Protein desaturase (T117R/G188L/D280K) that, in addition to introducing a double bond into stearate to produce oleate, performed an additional round of oxidation to convert oleate to a trans allylic alcohol acid. To determine the contributions of each mutation, in this work we generated individual castor desaturase mutants carrying residue changes corresponding to those in the triple mutant and investigated their catalytic activities. We observed that T117R, and to a lesser extent D280K, accumulated a novel product, namely erythro-9,10-dihydroxystearate, that we identified via its methyl ester through gas chromatography-mass spectrometry and comparison with authentic standards. The use of 18O2 labeling showed that the oxygens of both hydroxyl moieties originate from molecular oxygen rather than water. Incubation with an equimolar mixture of 18O2 and 16O2 demonstrated that both hydroxyl oxygens originate from a single molecule of O2, proving the product is the result of dioxygenase catalysis. Using prolonged incubation, we discovered that wild-type castor desaturase is also capable of forming erythro-9,10-dihydroxystearate, which presents a likely explanation for its accumulation to ∼0.7% in castor oil, the biosynthetic origin of which had remained enigmatic for decades. In summary, the findings presented here expand the documented constellation of di-iron enzyme catalysis to include a dioxygenase reactivity in which an unactivated alkene is converted to a vicinal diol.

中文翻译：

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蓖麻硬脂酰-ACP去饱和酶可以通过双加氧酶化学合成邻苯二甲酸。

在以前的工作中，我们鉴定了蓖麻（蓖麻）硬脂酰-酰基载体蛋白去饱和酶（T117R / G188L / D280K）的三重突变体，该突变体除了将双键引入硬脂酸酯中以产生油酸酯外，还进行了另一轮氧化将油酸酯转化为烯丙基醇酸 为了确定每个突变的贡献，在这项工作中，我们产生了单独的蓖麻去饱和酶突变体，其突变体具有对应于三重突变体的残基变化，并研究了其催化活性。我们观察到，T117R和较小的D280K积累了一种新产物，即9,10-二羟基硬脂酸赤藓酯，我们通过气相色谱-质谱联用它的甲酯鉴定出了该产物，并与真实标准品进行了比较。18O2标记的使用表明，两个羟基部分的氧均来自分子氧，而不是水。用等摩尔的18O2和16O2混合物温育表明，两个羟基氧均起源于一个单一的O2分子，证明了该产物是双加氧酶催化的结果。使用长时间的孵育，我们发现野生型蓖麻去饱和酶也能够形成9,10-二羟基硬脂酸赤藓酯，这可能解释了蓖麻去饱和酶在蓖麻油中的累积至〜0.7％，而蓖麻油的生物合成来源仍然是不可思议的。几十年。总而言之，这里提出的发现扩展了已记录的双铁酶催化的构象，使其涵盖了一种双加氧酶反应性，其中未活化的烯烃被转化为邻二醇。