MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites

MAD experiments attempting to solve the structure of 5-aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn2+ ion by Pb2+ ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg cocrystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg cocrystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg2+ had substituted for the same Zn2+ cofactor ion as had Pb2+, a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt2+ ions were found to bind at the same place in the structure. The refined structures of the P-band the Hg-complexed enzymes are presented at 2.5 and 3.0 Angstrom resolution, respectively.