Structure, Mechanism and Nickel Metallocenter Assembly of Lactate Racemase

该奖项将资助密歇根州立大学的Robert P.Hausinger教授和Jian Hu教授研究与乳酸生物合成有关的酶的结构和功能。这项研究的重点是乳酸消旋酶，它在一个看似简单但在化学上具有挑战性的反应中，将两种形式的乳酸相互转化。从这些络合物中获得的信息可用于其他依赖于镍金属中心的酶的研究。这项研究培训博士后研究人员使用质谱学、结构、光谱和突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以及查尔斯·德鲁科学学者计划、增加科学多样性和教育机会计划(IDEAS)和暑期研究机会计划(SROP)中代表性较低的本科生进行互动。此外，从这个项目中获得的信息被纳入正在进行的名为“生物学中的金属”、“蛋白质结构、功能和设计”和“集成微生物生物学”的研究生课程中。这个项目研究了最近发现的吡啶-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，它作为(SCS)Ni(II)钳形络合物与镍配位，并与乳酸外消旋酶Lys184共价结合。这种辅因子的合成需要辅助蛋白LARB、LARC和LARE，这些蛋白的作用尚不明确。计划中的分析包括通过质谱学研究蛋白质结合和游离中间体，通过X射线结晶学阐明这些蛋白质在不同状态下的结构，在催化过程中对LARA中间体进行分光光度分析和生物物理分析，以及通过提供适当的辅助因子来体外重现生物合成途径。这项研究有望扩大对镍生物化学的理解，并阐明氧化还原化学的机理细节，涉及一种拴系的、含镍的烟酸辅因子。(SCS)Ni(II)辅因子代表了无机化学家所熟知的第一个钳形络合物的生物例子。

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.