The overall purpose of this proposal is to develop new drug targets against the biosynthesis of the cell wall of Mycobacterium tuberculosis. One of the components of the wall, galactofuran, is an integral unit of the polymer arabinogalactan which links together the two essential layers of the mycobacterial cell wall, peptidoglyca and mycolic acids. Evidence, involving the mechanism of action of a know drug and the isolation of a temperature sensitive mutant, demonstrates that inhibition of the biosynthesis of arabinogalactan will stop mycobacterial cell growth and will probably result in cell death. As part of the parent grant, Dr McNeils' group has cloned and expressed UDP-galactopyranose mutase, the donor of galactofuranosyl residues.
The aims of this Fogarty International Research Collaboration Award (FIRCA) are to obtain the crystal structure of this enzyme which will dramatically enhance the Specific Aim of the parent grant to determine its mechanism of action. Additional aims of this grant are to obtain the crystal structure of UDP-galactopyranose mutase bound to substrate and substrate analogues and to obtain its structure after sited-directed mutagenesis. The foreign collaborator, Dr. James Naismith (University of St. Andrews, Scotland) has already crystallized the Escherichia coli and has small crystals of the Klebsiella pneumonia UDP-galactopyranose mutases. Using synchrotron radiation, Dr. Naismith has collected complete x-ray data on crystals of the E. coli enzymes to a resolution of 2.9 Å (space group P21, unit cell dimensions of a = 71.12 angstrom, b = 58.42 angstrom, c = 97.89 angstrom, alpha = gamma = 90.0, beta = 96.38_). Dr. Naismith's group has identified a platinum derivative of the E. coli enzyme using heavy metal soaking experiments and crystallized selenomethionine E. coli UDP-galactopyranose mutase.
