Structure, Mechanism and Nickel Metallocenter Assembly of Lactate Racemase

有了这个奖项，化学系的生命过程化学项目将资助密歇根州立大学的Robert P. Hausinger和Jian Hu教授，以表征乳酸生物合成中涉及的酶的结构和功能。这项研究的重点是乳酸消旋酶，它能将两种形式的乳酸相互转化，反应看似简单，但在化学上具有挑战性。从这些配合物中获得的信息可用于其他依赖镍金属中心的酶的研究。本研究培养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。这些人在高中荣誉科学项目中与有天赋的高中生互动，在查尔斯德鲁科学学者项目中与代表性不足的本科生互动，增加多样性和科学教育机会（IDEAS）项目，以及夏季研究机会项目（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”。本项目研究了最近发现的一种吡啶-3-硫酰胺-5-硫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形成（SCS）Ni（II）钳形复合物，并与乳酸消旋酶LarA的Lys184共价结合。这种辅助因子的合成需要作用尚未明确的辅助蛋白LarB、LarC和LarE。计划的分析包括通过质谱法研究蛋白质结合和游离中间体，通过x射线晶体学对这些蛋白质在不同状态下的结构进行阐明，在催化过程中对LarA中间体进行分光光度和生物物理分析，以及通过提供适当的辅助因子对生物合成途径进行体外重述。这项研究有望扩大对镍生物化学的理解，并阐明涉及系链的、含镍的烟酸辅助因子的氧化还原化学的机制细节。（SCS）Ni（II）辅因子是无机化学家所熟知的钳形复合物的第一个生物学例子。

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.