LAUSR

The MM2060 ( cobD) gene from Methanosarcina mazei strain Gö1 encodes a protein ( MmCobD) with L-threonine kinase (PduX) and L-threonine- O-3-phosphate decarboxylase (CobD) activities. In addition to the unexpected L-Thr kinase activity, MmCobD has an extended carboxy-terminal ( C-terminal) region annotated as a putative metal-binding zinc finger-like domain. Here we demonstrate that the C-terminus of MmCobD is a ferroprotein containing ~25 non-heme iron atoms per monomer of protein. The absence of the C-terminus substantially reduces, but does not abolish, enzymatic activities in vitro and in vivo. Single residue substitutions of C-terminal putative Fe-binding cysteinyl and histidinyl residues resulted in the loss of Fe and changes in enzyme activity levels. Salmonella enterica Δ pduX or Δ cobD strains were used as heterologous hosts to assess coenzyme B12 biosynthesis as a function of MmCobD variants. Some of the latter displayed 5-fold higher enzymatic activity in vitro and enhanced the growth rate of the S. enterica strains that synthesized them. Most of the MmCobD variants tested were 2- to 6-fold less active in vitro, and supported slow growth rates of the S. enterica strains that synthesized them; some substitutions abolished enzyme activity. MmCobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H2O2 and reduction by sodium dithionite. The presence of FeS clusters in MmCobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that MmCobD contains one or more diamagnetic [4Fe-4S]2+ center(s) that may play a structural or regulatory role.