Structural analysis of the protein shell of the propanediol utilisation metabolosome

Abstract

Propanediol metabolism occurs within a proteinaceous organelle in several bacterial species including Citrobacter freundii and Lactobacillus reuteri. The propanediol utilisation (Pdu) microcompartment shell is built from thousands of hexagonal shaped protein oligomers made from seven different types of protein subunits. In this Thesis, I investigate and analyse the structure and assembly of the bacterial microcompartment shell proteins. One of the shell proteins characterised in this work, PduT, has a tandem canonical bacterial microcompartment (BMC) repeat within the subunit and forms trimers with pseudo-hexagonal symmetry. This trimeric assembly forms a flat approximately hexagonally shaped disc with a central pore that is suitable for binding a 4Fe-4S cluster. The essentially cubic shaped 4Fe-4S cluster conforms to the threefold symmetry of the trimer with one free iron, the role of which could be to supply electrons to an associated microcompartment enzyme, PduS. The major shell protein PduB has a tandem permuted BMC repeat within the subunit and also forms trimers with pseudo-hexagonal symmetry. This shell protein closely resembles its homologous counterpart, EtuB; both possess three small pores formed within the subunits rather than a single pore at the centre of the pseudo-hexameric disc. The crystal structure of PduB provides insights into how substrates such as glycerol are able to use these pores as substrate channels. PduB appears to be able to pack within a sheet of PduA molecules, suggesting how the facet of the shell may be assembled. The higher order packing of shell proteins was investigated using PduA. Residues important for the packing of molecules into sheets were mutated and the effects on crystal morphology and on the shape of structures formed within the bacterial cell were assessed. PduA appears to assemble into straws in the bacterial cell and mutation of these residues has a profound influence on the structures produced.