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 co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal 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 Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 Å resolution, respectively.