Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase
Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase
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DOI：
10.1021/acs.biochem.8b00130
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发表时间：
2018-06-12
期刊：
影响因子：
2.9
通讯作者：
Silvaggi, Nicholas R.
中科院分区：
文献类型：
--
作者：
Han, Lanlan;Vuksanovic, Nemanja;Silvaggi, Nicholas R.
来自和田山链霉菌的PLP依赖性L-精氨酸羟化酶/脱氨酶MppP（SwMppP）参与L-精氨酸的生物合成，L-精氨酸是在几种非核糖体产生的肽类抗生素中发现的非蛋白质氨基酸。SwMppP仅使用PLP和分子氧来催化精氨酸的4电子氧化以形成2-氧代-4（S）-羟基-5-胍基戊酸和2-氧代-5-胍基戊酸的混合物。在过氧化氢酶的存在和不存在下的稳态动力学分析表明，一个分子的过氧化物形成的每一个分子的双氧在反应中消耗。此外，对于产生的每一分子2-氧代-4（S）-羟基-5-胍基戊酸，消耗两分子的分子氧，这表明4-羟基和2-酮基均衍生自水。通过使用O-[18]（2）或（H2O）-O-[18]运行反应并通过ESI-MS分析产物证实了这一点。仅当反应在（H2O）-O-[18]中进行时才观察到O-[18]的掺入。以2.2、1.9的分辨率测定了结合有L-精氨酸、2-氧代-4（S）-羟基-5-胍基戊酸或2-氧代-5-胍基戊酸的SwMppP的晶体结构。和1.8埃。结构数据表明，蛋白质的N-末端部分是无序的，除非底物或产物结合在活性位点，在这种情况下，它形成一个覆盖催化中心的有序螺旋。这一观察结果表明，N-末端螺旋可能在底物结合和/或催化中起作用。我们的N-末端变体的结构和动力学表征表明，N-末端是催化的关键。鉴于这一新的信息，我们已经完善了我们以前提出的机制SwMppP催化氧化L-精氨酸。
The PLP-dependent L-arginine hydroxylase/deaminase MppP from Streptomyces wadayamensis (SwMppP) is involved in the biosynthesis of L-enduracididine, a nonproteinogenic amino acid found in several nonribosomally produced peptide antibiotics. SwMppP uses only PLP and molecular oxygen to catalyze a 4-electron oxidation of Larginine to form a mixture of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid and 2-oxo-5-guanidinovaleric acid. Steady-state kinetics analysis in the presence and absence of catalase shows that one molecule of peroxide is formed for every molecule of dioxygen consumed in the reaction. Moreover, for each molecule of 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid produced, two molecules of dioxygen are consumed, suggesting that both the 4-hydroxy and 2-keto groups are derived from water. This was confirmed by running the reactions using either O-[18](2) or (H2O)-O-[18] and analyzing the products by ESI-MS. Incorporation of O-[18] was only observed when the reaction was performed in (H2O)-O-[18]. Crystal structures of SwMppP with L-arginine, 2-oxo-4(S)-hydroxy-5-guanidinovaleric acid, or 2-oxo-5-guanidinovaleric acid bound were determined at resolutions of 2.2, 1.9. and 1.8 angstrom, respectively. The structural data show that the N-terminal portion of the protein is disordered unless substrate or product is bound in the active site, in which case it forms a well-ordered helix that covers the catalytic center. This observation suggested that the N-terminal helix may have a role in substrate binding and/or catalysis. Our structural and kinetic characterizations of N-terminal variants show that the N-terminus is critical for catalysis. In light of this new information, we have refined our previously proposed mechanism of the SwMppP-catalyzed oxidation of L-arginine.