Structure, Mechanism and Nickel Metallocenter Assembly of Lactate Racemase

乳酸消旋酶的结构、机制及镍金属中心组装

• 批准号: 1516126
• 负责人: Robert Hausinger
• 金额: $ 54万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1516126&HistoricalAwards=false
• 关键词: Structure; Mechanism; Nickel; Metallocenter; Assembly

With this award, the Chemistry of Life Processes Program in the Division of Chemistry is funding Professors Robert P. Hausinger and Jian Hu of Michigan State University to characterize the structures and functions of enzymes involved in lactate biosynthesis. The enzyme that is the focus of this study, lactate racemase, interconverts two forms of lactic acid, in a deceptively simple, though chemically challenging, reaction. Information gained with these complexes may be used to inform other, studies of enzymes dependent upon nickel metal centers. This research trains postdoctoral researchers in the use of mass spectrometric, structural, spectroscopic, and mutagenesis studies to characterize the enzyme and its helper proteins. These individuals interact with gifted high school students in the High School Honors Science Program and underrepresented undergraduate students in the Charles Drew Science Scholars program, the Increasing Diversity and Education Access to Sciences (IDEAS) program, and the Summer Research Opportunities Program (SROP). Furthermore, information gained from this project is incorporated into ongoing graduate courses entitled "Metals in Biology," "Protein Structure, Function, and Design," and "Integrated Microbial Biology."This project examines the functional role and biosynthetic pathway of a recently identified pyridinium-3-thioamide-5-thiocarboxylic acid mononucleotide that coordinates nickel as a (SCS)Ni(II) pincer complex and is covalently bound to Lys184 of lactate racemase, LarA. Synthesis of this cofactor requires the accessory proteins LarB, LarC, and LarE of still undefined roles. Planned analyses include investigation of protein bound and free intermediates by mass spectrometry, structural elucidation of these proteins in their various states by x-ray crystallography, spectrophotometric and biophysical analyses of LarA intermediates during catalysis, and in vitro recapitulation of the biosynthetic pathway by supplying the appropriate cofactors. This research is expected to expand understanding of nickel biochemistry and elucidate mechanistic details of oxidation-reduction chemistry involving a tethered, nickel-containing, nicotinic acid cofactor. The (SCS)Ni(II) cofactor represents the first biological example of a pincer complex, well known to inorganic chemists.

有了这个奖项，化学系的生命过程化学项目正在资助密歇根州立大学的Robert P. Hausinger和Jian Hu教授，以表征参与乳酸生物合成的酶的结构和功能。乳酸消旋酶是这项研究的重点，它可以在一个看似简单但具有化学挑战性的反应中将两种形式的乳酸相互转化。从这些复合物中获得的信息可用于通知其他依赖于镍金属中心的酶的研究。这项研究训练博士后研究人员使用质谱，结构，光谱和诱变研究来表征酶及其辅助蛋白。这些人与高中荣誉科学计划中有天赋的高中生和查尔斯·德鲁科学学者计划中代表性不足的本科生互动，增加多样性和教育科学（IDEAS）计划和夏季研究机会计划（SROP）。此外，从这个项目中获得的信息被纳入正在进行的研究生课程，题为“生物学中的金属”，“蛋白质结构，功能和设计”和“综合微生物生物学”。“该项目研究了最近鉴定的吡啶鎓-3-硫代酰胺-5-硫代羧酸单核苷酸的功能作用和生物合成途径，该单核苷酸与镍配合为（SCS）Ni（II）钳形络合物，并与乳酸消旋酶LarA的Lys 184共价结合。这种辅助因子的合成需要辅助蛋白LarB、LarC和LarE，其作用尚不明确。计划的分析包括研究蛋白质结合和游离中间体的质谱法，这些蛋白质在其各种状态的结构阐明的X-射线晶体学，分光光度和生物物理分析的LarA中间体在催化过程中，并在体外重演的生物合成途径，通过提供适当的辅因子。这项研究有望扩大镍生物化学的理解，并阐明涉及拴系，含镍，烟酸辅因子的氧化还原化学的机理细节。（SCS）Ni（II）辅因子代表了无机化学家熟知的钳形络合物的第一个生物学实例。