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.
