The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis.
The role of conserved residues in Fdc decarboxylase in prenylated flavin mononucleotide oxidative maturation, cofactor isomerization, and catalysis.
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DOI：
10.1074/jbc.ra117.000881
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发表时间：
2018-02-16
期刊：
影响因子：
--
通讯作者：
Leys D
中科院分区：
文献类型：
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作者：
Bailey SS;Payne KAP;Fisher K;Marshall SA;Cliff MJ;Spiess R;Parker DA;Rigby SEJ;Leys D
可逆脱羧酶的UbiD家族作用于芳香族、杂芳香族和不饱和脂肪酸，并利用异戊二烯化黄素单核苷酸（prFMN）作为辅因子，与酶活性位点中的保守Glu-Arg-Glu/Asp离子网络相邻结合。有人提出，UbiD激活需要氧化成熟的辅因子，其中两个不同的异构体，prFMNketimine和prFMNiminium，已被观察到。也有人提出，只有prFMNiminium形式与催化有关，这需要底物和辅因子之间的瞬时环加成。使用尼日尔曲霉Fdc 1作为模型系统，我们发现，异构化的prFMNiminium prFMNketimine是一个光依赖性的过程，在很大程度上是独立的Glu 277-Arg 173-Glu 282网络，并伴随着不可逆的活性损失。另一方面，有效的催化高度依赖于完整的Glu-Arg-Glu网络，因为只有Glu → Asp取代保留活性。令人惊讶的是，形成prFMNiminium物质的氧化成熟仅对于R173 A变体受到严重影响。总之，prFMN的不寻常的不可逆异构化是光依赖性的，并且可能通过高能中间体进行，但不依赖于Glu-Arg-Glu网络。我们的结果从诱变，晶体学，光谱学和动力学实验表明一个明确的作用，谷氨酸-精氨酸-谷氨酸网络在催化和氧化成熟。
The UbiD family of reversible decarboxylases act on aromatic, heteroaromatic, and unsaturated aliphatic acids and utilize a prenylated flavin mononucleotide (prFMN) as cofactor, bound adjacent to a conserved Glu–Arg–Glu/Asp ionic network in the enzyme's active site. It is proposed that UbiD activation requires oxidative maturation of the cofactor, for which two distinct isomers, prFMNketimine and prFMNiminium, have been observed. It also has been suggested that only the prFMNiminium form is relevant to catalysis, which requires transient cycloaddition between substrate and cofactor. Using Aspergillus niger Fdc1 as a model system, we reveal that isomerization of prFMNiminium to prFMNketimine is a light-dependent process that is largely independent of the Glu277–Arg173–Glu282 network and accompanied by irreversible loss of activity. On the other hand, efficient catalysis was highly dependent on an intact Glu–Arg–Glu network, as only Glu → Asp substitutions retain activity. Surprisingly, oxidative maturation to form the prFMNiminium species is severely affected only for the R173A variant. In summary, the unusual irreversible isomerization of prFMN is light-dependent and probably proceeds via high-energy intermediates but is independent of the Glu–Arg–Glu network. Our results from mutagenesis, crystallographic, spectroscopic, and kinetic experiments indicate a clear role for the Glu–Arg–Glu network in both catalysis and oxidative maturation.