Structural Determinants of Flavin Dynamics in a Class B Monooxygenase
Structural Determinants of Flavin Dynamics in a Class B Monooxygenase
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DOI：
10.1021/acs.biochem.0c00783
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发表时间：
2020-12-08
期刊：
影响因子：
2.9
通讯作者：
Tanner, John J.
中科院分区：
文献类型：
--
作者：
Campbell, Ashley C.;Robinson, Reeder;Tanner, John J.
鸟氨酸羟化酶SidA是一种B类黄素单加氧酶，在烟曲霉中催化含羟酸铁载体生物合成的第一步。SidA的晶体学研究表明，在催化循环过程中，FAD在外态和在态之间经历了剧烈的构象变化。我们通过探测Met101的功能来深入了解B类单加氧酶中黄素运动的起源和目的，Met101是一种与in FAD的嘧啶环接触的残基。稳态动力学测量表明，突变变体M101A的周转率降低了25倍。利用稳态前动力学测量、pH谱和溶剂动力学同位素效应测量分离出导致稳态活性降低的微观步骤。该数据与机制最后一步的瓶颈一致，该步骤涉及黄素脱水和羟基-鸟氨酸和NADr的释放。测定了静息状态下M101A与NADr络合的晶体结构。静息酶结构与野生型SidA相似，与M101A一致，表现出NADPH对黄素还原的正常动力学和对NADPH的野生型亲和力。相反，M101A-NADP（+）复合物的结构出乎意料地显示FAD采用out构象，可能代表一种停滞构象，这是导致慢动力学的原因。总之，我们的数据支持先前的建议，即在B类黄素单加氧酶中FAD构象从内到外变化的一个目的是排出废NADP(+)，为新的催化循环做准备。
The ornithine hydroxylase known as SidA is a class B flavin monooxygenase that catalyzes the first step in the biosynthesis of hydroxamate-containing siderophores in Aspergillus fumigatus. Crystallographic studies of SidA revealed that the FAD undergoes dramatic conformational changes between out and in states during the catalytic cycle. We sought insight into the origins and purpose of flavin motion in class B monooxygenases by probing the function of Met101, a residue that contacts the pyrimidine ring of the in FAD. Steady-state kinetic measurements showed that the mutant variant M101A has a 25-fold lower turnover number. Pre-steady-state kinetic measurements, pH profiles, and solvent kinetic isotope effect measurements were used to isolate the microscopic step that is responsible for the reduced steady-state activity. The data are consistent with a bottleneck in the final step of the mechanism, which involves flavin dehydration and the release of hydroxy-Lornithine and NADr. Crystal structures were determined for M101A in the resting state and complexed with NADr. The resting enzyme structure is similar to that of wild-type SidA, consistent with M101A exhibiting normal kinetics for flavin reduction by NADPH and wild-type affinity for NADPH. In contrast, the structure of the M101A-NADP(+) complex unexpectedly shows the FAD adopting the out conformation and may represent a stalled conformation that is responsible for the slow kinetics. Altogether, our data support a previous proposal that one purpose of the FAD conformational change from in to out in class B flavin monooxygenases is to eject spent NADP(+) in preparation for a new catalytic cycle.