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

Abstract

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 ΔpduX or ΔcobD strains were used as heterologous hosts to assess coenzyme B 12 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 H 2 O 2 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.

Genome Scale Constraint-Based Metabolic Reconstruction of Propionibacterium Freudenreichii DSM 20271

“…On the other hand, one of the two enzymatic steps for the synthesis of (R)-1aminopropan-2-yl-phosphate (L-threonine 3-O-phosphate decarboxylase; EC: 4.1.1.81) has already been predicted by genome annotation data. We were also able to identify RM25_1014 as the putative encoder of L-threonine kinase (EC: 2.7.1.177) with 33.5% similarity with the bifunctional L-threonine kinase/L-threonine-O-3-phosphate decarboxylase (MM_2060) of Methanosarcina mazei Gö1 70 . Filling this gap accordingly enabled the vitamin B12 pathway to carry ux during FBA simulation.…”

Biochemical and genetic examination of two aminotransferases from the hyperthermophilic archaeon Thermococcus kodakarensis

“…These observations suggest that the physiological roles of members of G4 and G7 are related to Phe/Tyr and His biosynthesis, respectively. The members of G8 from Thermococcales have not been characterized, but display 30% identity to Thr decarboxylase (encoded by MM2060) from Methanosarcina mazei (Tavares et al, 2018(Tavares et al, , 2019 and are clustered with genes related to cobalamin salvage. By contrast, members of G1, G2, G3, G5, and G6 did not show a tendency to be included in a particular biosynthesis operon or gene cluster.…”