X-ray Analysis of Lead(II) Binding to Haloferax volcanii Malate Synthase.

Elucidation of protein structures at the atomic level using X-ray crystallography is an effective technique for studying metabolic enzymes and defining binding interactions of small molecules and ions. Our research centers on the structure of the malate synthase isoform H (MSH) from Haloferax volcanii, which is a key enzyme in the glyoxylate pathway of cellular metabolism and allows this organism to integrate two carbon compounds for anabolic biosynthetic reactions. We have collected X-ray diffraction data from a protein crystal soaked in a solution containing lead(II) acetate. This heavy atom derivative provided phasing information using the single isomorphous replacement with anomalous scattering (SIRAS) method to determine the structure of the native enzyme, but this structure has not previously been analyzed and fully refined. Here we report the iterative model-building and refinement of this structure at 2.1Å resolution to an overall R-value of 0.1831 and an R-free of 0.2167. This structure allows a detailed analysis of lead(II) ion binding to the protein with implications for lead toxicity and inhibition of the enzyme. In addition to the displacement of the required magnesium ion and accompanying distortions in the local vicinity of the active site, we find three additional binding sites for lead ions. Strong peaks are observed at these lead binding sites in anomalous difference Fourier maps, and there are very high electron-density peaks in the 2Fo-Fc map at these four locations. Lead binding at intersubunit contacts may explain the increased resolution of X-ray diffraction from this derivative versus the native protein. [ABSTRACT FROM AUTHOR]