Correction to oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.
Correction to oxygen-independent alkane formation by non-heme iron-dependent cyanobacterial aldehyde decarbonylase: investigation of kinetics and requirement for an external electron donor.
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通过非血红素铁依赖性蓝藻醛脱羰酶校正不依赖于氧的烷烃形成：动力学研究和外部电子供体的需求。
DOI：
10.1021/bi300837j
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发表时间：
2012-07-17
期刊：
影响因子：
2.9
通讯作者：
Marsh EN
中科院分区：
文献类型：
--
作者：
Eser BE;Das D;Han J;Jones PR;Marsh EN
在我们最初的出版物中，我们将cAD催化的脱羰基反应表征为不依赖于氧。支持这一观点的证据来自于这样一个事实，即反应不正式涉及底物的氧化，并且我们在厌氧条件下观察到比在有氧缓冲液中更高的周转次数。进一步的支持来自H2 18 O标记实验，表明甲酸盐中的氧来自水，而不是分子氧，以及烷烃的形成似乎没有消耗氧化反应所必需的NADH。然而，在发表之后，进一步的实验使我们得出结论，我们不能排除我们观察到的活性是由于反应缓冲液中的痕量氧的可能性。建立反应对分子氧的依赖性的困难，部分是由于在需氧或厌氧条件下酶的活性很低。我们已经发现，常规用于从生物化学反应中除去氧的氧洗涤系统，例如连二亚硫酸钠、葡萄糖氧化酶/葡萄糖和原儿茶酸双加氧酶/原儿茶酸盐，使用我们描述的测定条件，即使当大量过量包括时，仍然导致高水平的活性。尽管这些观察结果支持我们最初的断言，即不涉及氧气，但当在能够将氧气浓度维持在非常低的浓度（即< 0.5 ppm）（我们在最初的研究中无法获得）的厌氧室中进行测定时，我们观察到非常少的活性。虽然18 O标记实验应该已经确定了分子氧的参与，分析的数据是复杂的非酶交换18 O到醛底物从H2 18 O的背景速率。进行适当的对照以解释该背景反应，但是如果酶本身在反应之前显著增加18 O交换成醛的速率，则从实验得出的结论可能无效。我们目前正在进行进一步的实验，以澄清反应中对氧气和还原当量的要求，以解决这些结果中的差异。同时，鉴于这种差异，我们得出结论，我们不能明确排除分子氧参与cAD催化的反应。如果需要分子氧，则该酶对O2的表观Km必须非常低。
In our original publication, we characterized the decarbon-ylation reaction catalyzed by cAD as being independent of oxygen. Evidence to support this view derives from that fact that the reaction does not formally involve the oxidation of the substrate and that we observed a higher number of turnovers under anaerobic conditions than in aerobic buffers. Further support came from H2 18O labeling experiments that indicated that the oxygen in formate was derived from water, rather than molecular oxygen, and the fact that the formation of alkanes did not appear to consume NADH, which would be necessary for an oxidative reaction. However, subsequent to publication, further experiments have led us to conclude that we cannot exclude the possibility that the activity we observed was due to trace amounts of oxygen in the reaction buffer. The difficulties in establishing the dependence of the reaction on molecular oxygen stem, in part, from the very low activity of the enzyme under either aerobic or anaerobic conditions. We have found that oxygen scrubbing systems that are routinely employed to scavenge oxygen from biochemical reactions, such as sodium dithionite, glucose oxidase/glucose, and protocatechuate dioxygenase/protocatechuate, still result in high levels of activity using the assay conditions we describe, even when included in large excess. Although these observations support our initial assertion that oxygen was not involved, when the assays were performed in an anaerobic chamber capable of maintaining oxygen concentrations at very low concentrations, ie,< 0.5 ppm (which was not available to us in our original investigations), we observed very little activity. Although the 18O labeling experiment should have identified the involvement of molecular oxygen, analysis of the data was complicated by a background rate of nonenzymatic exchange of 18O into the aldehyde substrate from H2 18O. Appropriate controls were performed to account for this background reaction, but if the enzyme itself significantly increased the rate of exchange of 18O into the aldehyde prior to reaction, the conclusions drawn from the experiment may not be valid. We are currently conducting further experiments to clarify the requirement for oxygen and reducing equivalents in the reaction to resolve the discrepancy in these results. In the meantime, given this discrepancy, we conclude that we cannot unambiguously rule out the involvement of molecular oxygen in the cAD-catalyzed reaction. If molecular oxygen is required, the enzyme must have a very low apparent Km for O2.
影响因子：
2.9
作者：
Eser, Bekir E.;Das, Debasis;Han, Jaehong;Jones, Patrik R.;Marsh, E. Neil G.
通讯作者：
Marsh, E. Neil G.