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Streptomyces wadayamensis MppP is a PLP-Dependent Oxidase, Not an Oxygenase
Biochemistry ( IF 2.9 ) Pub Date : 2018-02-23 00:00:00 , DOI: 10.1021/acs.biochem.8b00130
Lanlan Han 1 , Nemanja Vuksanovic 1 , Sarah A. Oehm 1 , Tyler G. Fenske 1 , Alan W. Schwabacher 1 , Nicholas R. Silvaggi 1
Affiliation  

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 l-arginine 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 [18]O2 or H2[18]O and analyzing the products by ESI-MS. Incorporation of [18]O was only observed when the reaction was performed in H2[18]O. 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 Å, 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.

中文翻译:

wadayamensis链霉菌MppP是PLP依赖性氧化酶,而不是氧化酶

来自瓦达链霉菌(SwMppP)的PLP依赖的1-精氨酸羟化酶/脱氨酶MppP参与了1- enduracididine的生物合成,L- enduracididine是在几种非核糖体生产的多肽抗生素中发现的非蛋白氨基酸。SwMppP仅使用PLP和分子氧来催化l的4电子氧化-精氨酸形成2-氧代-4(S)-羟基-5-胍基新戊酸和2-氧代-5-胍基新戊酸的混合物。有和没有过氧化氢酶的稳态动力学分析表明,反应中消耗的每个双氧分子均形成一个过氧化物分子。此外,对于每个产生的2-氧代-4(S)-羟基-5-胍基新戊酸分子,消耗了两个分子的双氧,这表明4-羟基和2-酮基均来自水。通过使用[18] O 2或H 2 [18] O进行反应并通过ESI-MS分析产物,可以证实这一点。仅在反应在H 2中进行时观察到[18] O的结合[18]O.以2.2、1.9的分辨率测定结合有1-精氨酸,2-氧代-4(S)-羟基-5-胍基新戊酸或2-氧代-5-胍基新戊酸的SwMppP的晶体结构。和1.8Å。结构数据表明,除非底物或产物结合在活性位点上,否则蛋白质的N端部分是无序的,在这种情况下,它会形成排列整齐的螺旋,覆盖催化中心。该观察结果表明,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末端对于催化至关重要。根据这一新信息,我们完善了我们先前提出的SwMppP催化的1-精氨酸氧化机理。
更新日期:2018-02-23
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