The 2.7 Å resolution structure of the catalytic domain of the dihydrolipoamide succinyltransferase from Escherichia coli in complex with coenzyme A and the 1.45 Å resolution structure of murine macrophage migration inhibitory factor in complex with phenylacetylenepyruvate

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2005

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Golubkov, Pavel Aleksandrovich

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Abstract

The work presented in this dissertation includes structural analysis of liganded forms of the truncated catalytic domain of the trimeric form of the dihydrolipoamide succinyltransferase from E. coli (E2oCD) and the macrophage migration inhibitory factor (MIF) from mouse. Part one of this dissertation deals with the analysis of the 2.7 Å resolution structure of E2oCD in complex with coenzyme A. The structure of the native E2oCD was solved in this laboratory previously. This project resulted in the crystal structure of the E2oCD·CoA complex obtained by molecular replacement and the resulting analysis of the chemical nature of the complex. Extensive comparisons were made with the previously reported structure of E2p from A. vinelandii, in complex with CoA and DHLA, as well as with the predicted ligand location based on computer models for E2oCD. Residues in the active site cleft are implicated in providing substrate-binding specificity. CoA is observed in the extended “IN” conformation, consistent with the unoccupied DHLA binding site. The second project reported here involves the study of MIF, a cytokine with unusual tautomerase activity. MIF is an important immunoregulatory protein with a potential to mediate inflammatory diseases. The details of the tautomerase activity MIF and its importance in MIF's biological functions remain elusive. This project resulted in a high-resolution X-ray structure of mouse MIF at 1.45 Å resolution in complex with an inhibitor phenylacetylenepyruvate (PAP). Well-defined electron density for Glu-16’ from a neighboring trimer was observed in one of the three active sites, with density for an inhibitor covalently bound to Pro-1 observed in the other two active sites. An analysis of the apparent covalent bond between the protein and the inhibitor and the potential ramifications for the understanding of the chemistry of formation of the adduct are discussed. The observed electron density was not consistent with the predicted Michael addition product.

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