MppP: The First PLP-Dependent Hydroxylase/Deaminase

MppP：第一个 PLP 依赖性羟化酶/脱氨酶

• 批准号: 1606842
• 负责人: Nicholas Silvaggi
• 金额: $ 50万
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1606842&HistoricalAwards=false
• 关键词: MppP; First; PLP; Dependent; Hydroxylase

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Nicholas Silvaggi from University of Wisconsin-Milwaukee to study how protein structure affects its function. In particular he is studying an enzyme known as MppP which reacts with molecular oxygen to add a hydroxyl group into the amino acid arginine. What is most interesting about MppP is that, while its structure closely resembles that of several other related proteins, its chemistry is very different. By studying MppP the PI is learning how to make educated guesses about the functions of the many uncharacterized proteins uncovered in genome sequencing. In addition, understanding how MppP works is improving understanding of natural products biosynthesis and the processes by which existing enzyme structures are adapted to perform new functions. As part of this project the PI is also developing the PX Lab experience, a program designed to give exceptional students from area high schools an immersive experience in modern structural biology research. The students are working as a team with their teachers, and supervised by the PI and his graduate students, to clone, express, purify, crystallize, and determine the structure of a fluorescent protein. In this way, the research being done in the lab is also training teachers and tomorrow's scientists.The non-proteinogenic amino acid L-enduracididine (L-End) is a component of a number of bacterially-produced natural products. The pathway for the production of L-End from arginine is thought to involve some unique enzymatic activities, but the reactions catalyzed by the three biosynthetic enzymes, MppP, MppQ and MppR, are unknown. Recent findings show that MppP is a previously unknown class of oxygenase that requires only PLP and molecular oxygen to insert an oxygen atom into an unactivated C-H bond. This is an unprecedented activity for a PLP-dependent enzyme. The objective of this work is to understand how MppP catalyzes this reaction and which structural features account for its unusual activity.Ppre-steady state enzyme kinetics, together with kinetic isotope effects, NMR spectroscopy, and mass spectrometry are being used to probe the catalytic mechanism. Structural features of MppP required for its hydroxylation reaction is being identified by X-ray crystallographic and enzymes kinetics studies of mutant forms of MppP, both alone and in complexes with ligands. The outcome of the research is detailed mechanistic information about how MppP carries out its reaction, which is helping understand how evolution has modified the Type I aminotransferase fold to perform a new catalytic function. These outcomes are expanding knowledge of PLP-dependent enzymes, specifically, of enzyme structure-function relationships, as well as improving the accuracy of protein function predictions.

有了这个奖项，化学部的生命过程化学项目资助威斯康星大学密尔沃基分校的Nicholas Silvaggi博士研究蛋白质结构如何影响其功能。特别是，他正在研究一种名为MppP的酶，这种酶与分子氧反应，将羟基加入氨基酸精氨酸中。关于MppP最有趣的是，虽然它的结构与其他几种相关蛋白质非常相似，但它的化学性质却非常不同。通过研究MppP，PI正在学习如何对基因组测序中发现的许多未表征蛋白质的功能进行有根据的猜测。此外，了解MppP的工作原理有助于提高对天然产物生物合成以及现有酶结构适应新功能的过程的理解。作为该项目的一部分，PI还开发了PX实验室体验，该项目旨在为来自地区高中的优秀学生提供现代结构生物学研究的沉浸式体验。学生们与他们的老师组成一个团队，在PI和他的研究生的监督下，克隆，表达，纯化，结晶和确定荧光蛋白的结构。通过这种方式，在实验室进行的研究也在培训教师和未来的科学家。非蛋白质氨基酸L-精氨酸（L-End）是许多细菌产生的天然产物的组成部分。精氨酸生成L-末端的途径被认为涉及一些独特的酶活性，但由三种生物合成酶MppP、MppQ和MppR催化的反应尚不清楚。最近的研究表明，MppP是一种以前未知的加氧酶，只需要PLP和分子氧插入一个氧原子到未活化的C-H键。这对于PLP依赖性酶来说是前所未有的活性。这项工作的目的是了解如何MppP催化这一反应，并解释其不寻常的activity.Ppre-steady状态酶动力学，连同动力学同位素效应，NMR光谱和质谱的结构特征正在被用来探测催化机制。其羟基化反应所需的MppP的结构特征正在确定的X-射线晶体学和酶动力学研究的突变形式的MppP，单独和与配体的复合物。这项研究的结果是关于MppP如何进行其反应的详细机制信息，这有助于理解进化如何修改I型转氨酶折叠以执行新的催化功能。这些结果扩展了对PLP依赖性酶的了解，特别是酶结构-功能关系，以及提高蛋白质功能预测的准确性。