The l-Thr Kinase/l-Thr-Phosphate Decarboxylase (CobD) Enzyme from Methanosarcina mazei Gö1 Contains Metallocenters Needed for Optimal Activity.

The MM2060 ( cobD ) gene from Methanosarcina mazei strain Gö1 encodes a protein ( Mm CobD) with l-threonine kinase (PduX) and l-threonine- O -3-phosphate decarboxylase (CobD) activities. In addition to the unexpected l-Thr kinase activity, Mm CobD 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 Mm CobD 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 and Δ cobD strains were used as heterologous hosts to assess coenzyme B ₁₂ biosynthesis as a function of 17 Mm CobD variants tested. 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 Mm CobD variants tested were up 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. Mm CobD exhibited an ultraviolet-visible absorption spectrum consistent with [4Fe-4S] clusters that appeared to be susceptible to oxidation by H ₂ O ₂ and reduction by sodium dithionite. The presence of FeS clusters in Mm CobD was corroborated by electron paramagnetic resonance and magnetic circular dichroism studies. Collectively, our results suggest that Mm CobD contains one or more diamagnetic [4Fe-4S] ²⁺ center(s) that may play a structural or regulatory role.