Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, United States.
J Am Chem Soc. 2023 Sep 6;145(35):19256-19264. doi: 10.1021/jacs.3c04542. Epub 2023 Aug 23.
The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes.
细胞色素 P450 (CYP) AspB 参与二酮哌嗪 (DKP) aspergilazine A 的生物合成。色氨酸连接的二聚 DKP 生物碱是一个大型的天然产物家族，存在于许多物种中，具有广泛而通常有效的生物活性。AspB 形成 C-N 键的提议机制，以及相关 CYP 形成类似的 C-C 键的机制，都涉及使用铁氧中间物作为氧化剂来促进底物二聚化。在这里，稳态和瞬态动力学方法的平行应用揭示了一种截然不同的机制，涉及铁过氧化物物种作为主要氧化剂来引发 DKP 组装。单转换动力学同位素效应和底物类似物表明了可能的性质和位置。CYP-超氧化物反应性的直接观察合理化了 AspB 的非典型结果，并揭示了血红素酶中的一个新反应机制。
