Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.

中文翻译：

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底物结合可调节组蛋白 3-羟化酶的反应性，这是一种参与真菌生物发光的黄素蛋白单加氧酶。

最近显示真菌生物发光依赖于几种酶的独特氧依赖系统。然而，酶的身份并没有揭示这个过程的完整生化细节，因为这些酶与其他发光系统的酶没有相似之处，因此参与这个迷人过程的酶的特性仍然未知。在这里，我们描述了该途径中倒数第二个酶的特征，即来自发光真菌迈锡纳氯磷 (McH3H) 的组蛋白 3-羟化酶，该酶催化组蛋白转化为 3-羟基组蛋白。3-Hydroxyhispidin 在发光真菌中充当荧光素底物。McH3H 在大肠杆菌中异源表达并通过亲和层析纯化，产量为 100 毫克/升。发现 McH3H 是一种单组分单体 NAD(P)H 依赖的含 FAD 的单加氧酶，对 NADPH 有偏好。通过基于模型结构的定点诱变，产生了对 NADH 更有效的突变酶。除了确定调节辅因子特异性的残基外，这些工程变体还可能有助于开发新的基于组蛋白的生物发光应用。我们证实，向 McH3H 添加组氨酸导致形成 3-羟基组氨酸作为唯一的芳香族产物。快速动力学分析表明，NADPH 对黄素辅因子的减少受到了近 100 倍的 Hispidin 结合的促进。与其他 A 类黄素蛋白羟化酶相似，McH3H 不形成稳定的氢过氧黄素中间体。