Romero Elvira, Robinson Reeder, Sobrado Pablo
Department of Biochemistry, Virginia Polytechnic Institute and State University, Virginia, USA.
J Vis Exp. 2012 Mar 18(61):3803. doi: 10.3791/3803.
Aspergillus fumigatus siderophore A (SidA) is an FAD-containing monooxygenase that catalyzes the hydroxylation of ornithine in the biosynthesis of hydroxamate siderophores that are essential for virulence (e.g. ferricrocin or N',N",N'''-triacetylfusarinine C). The reaction catalyzed by SidA can be divided into reductive and oxidative half-reactions. In the reductive half-reaction, the oxidized FAD bound to Af SidA, is reduced by NADPH. In the oxidative half-reaction, the reduced cofactor reacts with molecular oxygen to form a C4a-hydroperoxyflavin intermediate, which transfers an oxygen atom to ornithine. Here, we describe a procedure to measure the rates and detect the different spectral forms of SidA using a stopped-flow instrument installed in an anaerobic glove box. In the stopped-flow instrument, small volumes of reactants are rapidly mixed, and after the flow is stopped by the stop syringe, the spectral changes of the solution placed in the observation cell are recorded over time. In the first part of the experiment, we show how we can use the stopped-flow instrument in single mode, where the anaerobic reduction of the flavin in Af SidA by NADPH is directly measured. We then use double mixing settings where Af SidA is first anaerobically reduced by NADPH for a designated period of time in an aging loop, and then reacted with molecular oxygen in the observation cell. In order to perform this experiment, anaerobic buffers are necessary because when only the reductive half-reaction is monitored, any oxygen in the solutions will react with the reduced flavin cofactor and form a C4a-hydroperoxyflavin intermediate that will ultimately decay back into the oxidized flavin. This would not allow the user to accurately measure rates of reduction since there would be complete turnover of the enzyme. When the oxidative half-reaction is being studied the enzyme must be reduced in the absence of oxygen so that just the steps between reduction and oxidation are observed. One of the buffers used in this experiment is oxygen saturated so that we can study the oxidative half-reaction at higher concentrations of oxygen. These are often the procedures carried out when studying either the reductive or oxidative half-reactions with flavin-containing monooxygenases. The time scale of the pre-steady-state experiments performed with the stopped-flow is milliseconds to seconds, which allow the determination of intrinsic rate constants and the detection and identification of intermediates in the reaction. The procedures described here can be applied to other flavin-dependent monooxygenases.
烟曲霉铁载体A（SidA）是一种含黄素腺嘌呤二核苷酸（FAD）的单加氧酶，它催化鸟氨酸的羟基化反应，该反应发生在对毒力至关重要的异羟肟酸铁载体（如铁载体菌素或N'，N''，N'''-三乙酰基镰刀菌素C）的生物合成过程中。SidA催化的反应可分为还原和氧化两个半反应。在还原半反应中，与烟曲霉SidA结合的氧化型FAD被烟酰胺腺嘌呤二核苷酸磷酸（NADPH）还原。在氧化半反应中，还原型辅因子与分子氧反应形成C4a-氢过氧化黄素中间体，该中间体将一个氧原子转移给鸟氨酸。在此，我们描述了一种使用安装在厌氧手套箱中的停流仪器来测量反应速率并检测SidA不同光谱形式的方法。在停流仪器中，少量反应物快速混合，当流动被停流注射器停止后，观察池中溶液的光谱变化随时间被记录下来。在实验的第一部分，我们展示了如何在单模式下使用停流仪器，直接测量NADPH对烟曲霉SidA中黄素的厌氧还原。然后我们使用双混合设置，在老化回路中首先用NADPH对烟曲霉SidA进行指定时间的厌氧还原，然后在观察池中与分子氧反应。为了进行这个实验，厌氧缓冲液是必需的，因为当仅监测还原半反应时，溶液中的任何氧气都会与还原型黄素辅因子反应并形成C4a-氢过氧化黄素中间体，该中间体最终会分解回氧化型黄素。这将不允许用户准确测量还原速率，因为酶会完全周转。当研究氧化半反应时，酶必须在无氧条件下还原，以便仅观察还原和氧化之间的步骤。本实验中使用的一种缓冲液是氧饱和的，这样我们可以在较高氧浓度下研究氧化半反应。这些通常是在用含黄素单加氧酶研究还原或氧化半反应时所进行的步骤。用停流进行的预稳态实验的时间尺度是毫秒到秒，这使得能够确定内在速率常数并检测和鉴定反应中的中间体。这里描述的方法可以应用于其他黄素依赖性单加氧酶。
