Distinct Roles for Two Trypsin-like Proteases in Magnetosome Biomineralization
The ability of living organisms to transform inorganic elements into insoluble crystalline structures is an underexplored theme in biology. A group of aquatic bacteria, called magnetotactic bacteria, produces chains of nanometer-sized magnetic crystals within their cells, allowing them to align in the earth’s geomagnetic fields. Understanding the mechanism for this process has become increasingly important as the demand for customized nanoparticles in medical and industrial applications has grown. In particular, the protein factors required to transform soluble iron into magnetite (Fe3O4) are likely to contain novel mechanisms for manipulating insoluble inorganic compounds.
This thesis describes the biochemical and genetic features of two such factors, MamO and MamE. A historical account is provided describing the discovery of magnetotactic bacteria, the development of Magnetospirillum magneticum AMB-1 as a model system and the identification of specific genes required to produce magnetite. This previous body of work led to the identification of MamO and MamE as predicted trypsin-like proteases that are required for biomineralization in AMB-1. Genetic, biochemical and structural studies reported here showed that the MamO protease domain is catalytically inactive and incapable of serine protease activity. Instead it has a novel di-histidine motif that participates in direct binding to transition metals. This motif is required for biomineralization in vivo, confirming that the MamO protein is a repurposed trypsin-like scaffold that promotes magnetite nucleation by binding directly to iron precursor atoms. Genomic and phylogenetic analysis of related serine proteases in other magnetotactic bacteria showed that the repurposing of trypsin-like proteases has occurred numerous times independently during the evolution of magnetosome formation. Also described is the observation that three biomineralization factors, MamE, MamO and MamP are proteolytically processed in AMB-1. MamE and MamO are both required for these proteolytic events, as are the predicted catalytic residues from MamE. However, consistent with its newly assigned pseudo-protease classification, the predicted MamO active site is dispensable. This suggested that MamE directly processes these targets in a manner that requires MamO. The proteolytic activity of MamE was reconstituted in vitro with a recombinant form of the protein. MamE cleaved a custom peptide substrate based on an in vivo cleavage site in MamO with positive cooperativity, and its auto-proteolytic activity could be stimulated by both substrates and peptides that bind to its regulatory domains. These enzymatic properties suggested that a switch-like regulatory mechanism modulated MamE-dependent proteolysis during biomineralization. This regulatory paradigm was confirmed by showing that both catalytically inactive and constitutively active alleles of mamE caused severe biomineralization defects in vivo.
Although the genes required for biomineralization were known previously, the molecular mechanisms by which each protein promotes magnetite synthesis had not been explored. The results of these studies define biochemical functions for two of the four factors required for magnetite nucleation in AMB-1. Furthermore, describing the evolutionary repurposing of a trypsin scaffold along with the phylogenetic description of its evolutionary history add broad biological interest to magnetosome research.