MamO Is a Repurposed Serine Protease that Promotes Magnetite Biomineralization through Direct Transition Metal Binding in Magnetotactic Bacteria

Abstract

Many living organisms transform inorganic atoms into highly ordered crystalline materials. An elegant example of such biomineralization processes is the production of nano-scale magnetic crystals in magnetotactic bacteria. Previous studies implicated the involvement of two putative serine proteases, MamE and MamO, during the early stages of magnetite formation in Magnetospirillum magneticum AMB-1. Here, using genetic analysis and X-ray crystallography, we show that MamO has a degenerate active site, rendering it incapable of protease activity. Instead, MamO promotes magnetosome formation through two genetically distinct, noncatalytic activities: activation of MamE-dependent proteolysis of biomineralization factors and direct binding to transition metal ions. By solving the structure of the protease domain bound to a metal ion, we identify a surface-exposed di-histidine motif in MamO that contributes to metal binding and show that it is required to initiate biomineralization in vivo. Finally, we find that pseudoproteases are widespread in magnetotactic bacteria and that they have evolved independently in three separate taxa. Our results highlight the versatility of protein scaffolds in accommodating new biochemical activities and provide unprecedented insight into the earliest stages of biomineralization.

Magnetite biomineralization in Magnetospirillum magneticum is regulated by a switch-like behavior in the HtrA protease MamE

“…The peptide-based substrate and ligands developed here shift MamE into the activated state in vitro, but the specific signals that activate the protein in vivo are not known. We previously showed that proteolysis of MamE, MamO and MamP requires the presence of MamO's Cterminal ion transporter domain, suggesting that manipulation of the solute environment in the magnetosome regulates MamE's activity (24). However, the transport cargo(es) of MamO have not been identified, making the prediction of potential activating solutes difficult.…”

Magnetite Biomineralization in Magnetospirillum magneticum Is Regulated by a Switch-like Behavior in the HtrA Protease MamE

“…For analysis of the autocleavage reaction products, a 1:10 dilution of each time point was separated on a 12% (v/v) acrylamide gel for immunoblotting. The MamE and MamP antibodies have been described previously (24). The anti-His 6 (Sigma), anti-FLAG (Sigma), anti-70 (Thermo Fisher), and anti-Strep-tag (Qiagen) antibodies were purchased from commercial sources.…”

“…We previously showed that proteolysis of MamE, MamO, and MamP requires the presence of the C-terminal ion transporter domain of MamO, suggesting that manipulation of the solute environment in the magnetosome regulates the activity of MamE (24). However, the transport cargo(s) of MamO have not been identified, making the prediction of potential activating solutes difficult.…”

“…Although MamO was also required for these proteolytic events, this effect did not require its predicted protease active site. Subsequent structural analysis showed that the protease domain of MamO was locked in an inactive state and incapable of catalysis, suggesting that it played a non-catalytic role in activating MamE (24).…”

Genomic study of a novel magnetotacticAlphaproteobacteriauncovers the multiple ancestry of magnetotaxis

“…Biomineralization of magnetic iron oxide and iron sulfide crystals by bacteria is a fascinating function whose underlying genetic determinism, molecular machinery, ecologic role and evolution have been the focus of an increasing number of investigations lately (Lefèvre et al, 2011;Lefèvre and Bazylinski, 2013;Hershey et al, 2016;Toro-Nahuelpan et al, 2016;Uebe and Schüler, 2016;Lin et al, 2017bLin et al, , 2018. To date, this ability has been observed in Gram-negative magnetotactic bacteria (MTB) only, which are commonly found at the oxic-anoxic interface (OAI) of any stratified freshwater or marine environment with an O 2 /redox gradient (Bazylinski and Frankel, 2004).…”