Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases
Evidence for Only Oxygenative Cleavage of Aldehydes to Alk(a/e)nes and Formate by Cyanobacterial Aldehyde Decarbonylases
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DOI：
10.1021/bi300912n
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发表时间：
2012-10-09
期刊：
影响因子：
2.9
通讯作者：
Bollinger, J. Martin, Jr.
中科院分区：
文献类型：
--
作者：
Li, Ning;Chang, Wei-chen;Bollinger, J. Martin, Jr.
蓝藻醛脱羰酶（AD）催化C-n脂肪醛转化为甲酸（HCO 2-）和相应的Cn-1烷（a/e）。先前对大肠杆菌（Ec）中产生的点状念珠藻（Np）AD的研究表明，这种表面上的水解反应实际上是一个隐藏的氧化还原加氧过程，其中一个O-原子从O-2并入甲酸盐，并且基于蛋白质的还原系统（NADPH，铁氧还蛋白和铁氧还蛋白还原酶; N/F/FR）提供O-2完全还原所需的所有四个电子。Marsh和同事的两个后续出版物[Das等人（2011）Angew. Chem.Int.Ed.50，7148-7152; Eser等人（2011）Biochemistry 50，10743-10750]报道了他们的Ec表达的Np和海洋原绿球藻（Prochlorococcus marinus，Pm）AD制剂通过不依赖于O-2的真正水解过程更快速地将醛转化为相同的产物，他们建议通过还原系统强制参与的瞬时底物还原进行（他们使用化学系统，NADH和吩嗪硫酸甲酯; N/PMS）。为了解决这一差异，我们重新检查了我们的两种AD直向同源物的制备，方法是（i）在存在和不存在O-2的情况下进行活性测定，以及（ii）O-18（2）和（H2O）-O-18同位素示踪实验，并直接质谱检测HCO 2产物。对于AD直向同源物（Np和Pm）、还原系统（基于蛋白质的和化学的）和底物（正庚醛和正十八醛）的多种组合，我们的制剂严格要求O-2的活性，并且不支持可检测的水解甲酸盐产生，尽管具有类似于或大于Marsh及其同事报道的催化活性。我们的研究结果，特别是O-18示踪实验，表明Marsh及其同事观察到的活性可能是由他们的测定中污染O-2引起的。明确重申的氧化反应的性质意味着，酶，最初指定为醛脱羰酶时，Cl-衍生的副产品被认为是一氧化碳，而不是甲酸，应重新指定为脱氢脱甲酰加氧酶（ADO）。
Cyanobacterial aldehyde decarbonylases (ADs) catalyze the conversion of C-n fatty aldehydes to formate (HCO2-) and the corresponding Cn-1 alk(a/e)nes. Previous studies of the Nostoc punctiforme (Np) AD produced in Escherichia coli (Ec) showed that this apparently hydrolytic reaction is actually a cryptically redox oxygenation process, in which one O-atom is incorporated from O-2 into formate and a protein-based reducing system (NADPH, ferredoxin, and ferredoxin reductase; N/F/FR) provides all four electrons needed for the complete reduction of O-2. Two subsequent publications by Marsh and co-workers [Das, et al. (2011) Angew. Chem. Int. Ed. 50, 7148-7152; Eser, et al. (2011) Biochemistry 50, 10743-10750] reported that their Ec-expressed Np and Prochlorococcus marinus (Pm) AD preparations transform aldehydes to the same products more rapidly by an O-2-independent, truly hydrolytic process, which they suggested proceeded by transient substrate reduction with obligatory participation by the reducing system (they used a chemical system, NADH and phenazine methosulfate; N/PMS). To resolve this discrepancy, we re-examined our preparations of both AD orthologues by a combination of (i) activity assays in the presence and absence of O-2 and (ii) O-18(2) and (H2O)-O-18 isotope-tracer experiments with direct mass-spectrometric detection of the HCO2- product. For multiple combinations of the AD orthologue (Np and Pm), reducing system (protein-based and chemical), and substrate (n-heptanal and n-octadecanal), our preparations strictly require O-2 for activity and do not support detectable hydrolytic formate production, despite having catalytic activities similar to or greater than those reported by Marsh and co-workers. Our results, especially of the O-18-tracer experiments, suggest that the activity observed by Marsh and co-workers could have arisen from contaminating O-2 in their assays. The definitive reaffirmation of the oxygenative nature of the reaction implies that the enzyme, initially designated as aldehyde decarbonylase when the Cl-derived coproduct was thought to be carbon monoxide rather than formate, should be redesignated as aldehyde-deformylating oxygenase (ADO).