Structural and biophysical investigations of [4Fe-4S] cluster coordinating proteins in malaria and herbicide biosynthesis

Abstract

Radical S-adenosylmethionine (RS) enzymes are the largest known enzyme superfamily, consisting of millions of reported sequences. RS enzymes use a [4Fe-4S] cluster to reductively cleave a S-adenosylmethionine (AdoMet) molecule to form a 5′-deoxyadenosine radical (5′-dAdo•). By using the 5′-dAdo•, RS enzymes catalyze a diverse portfolio of chemically difficult reactions. Cobalamin (Cbl)-dependent RS enzymes comprise a subfamily within the RS superfamily which employ both Cbl and 5′-dAdo• to catalyze reactions. Recently, two enzymes were identified as essential for the production of the herbicidal compound Albucidin: the RS enzyme AlsA and the Cbl-dependent RS enzyme AlsB. In this dissertation, we present our progress toward an X-ray crystallographic structure of AlsA and a cryogenic-electron microscopy (Cryo-EM) reconstruction of AlsB. The X-ray diffraction of AlsA crystals is low resolution, mosaic, and twinned, resulting in a 3.7 Å resolution electron density map with a disordered active site. Using Cryo-EM, a 7.12 Å resolution map of AlsB was generated. At 83.5 kDa in size, AlsB is small for Cryo-EM. The Cryo-EM map of AlsB closely resembles a closed conformation of the enzyme, a conformation that remains unobserved in the similar Cbl-dependent RS enzyme OxsB. Additionally, we describe an optimized procedure for the chemical reconstitution of AlsA suitable for structural studies. To assess the thermal stability of AlsA, a NanoDSF-based thermal shift assay was developed, demonstrating the presence of two protein populations. The two populations, hypothesized to be reconstituted and unreconstituted AlsA, react differently to the presence of dithiothreitol, a commonly used reductant for RS enzymes. Our findings suggest thermal shift assays can visualize heterogeneity in protein samples that otherwise could not be observed. Also, NanoDSF provides us with a high throughput method for determining suitable buffer conditions for structural studies for RS enzymes and other oxygen sensitive proteins. Finally, we report the second X-ray crystal structure of the essential lipocalin HAL from the malaria-causing parasite Plasmodium falciparum. Our crystal structure solved in the C2 space group shows a crystal lattice formed by repeated dodecamer rings. We identify chloride anion sites between two monomers, suggesting a role for chloride in the oligomerization of PfHAL. Altogether, this work provides a foundation for the structural investigation of Albucidin Biosynthesis and provides new information toward understand of the role of PfHAL in malaria.