Cobamides (Cbas) are synthesized by many archaea, but some aspects of Cba biosynthesis in these microorganisms remain unclear. Here, we demonstrate here that the ORF MM2060 in the archaeum Methanosarcina mazei strain Gö1 encodes a bifunctional enzyme with L-threonine-O-3-phosphate (L-Thr-P) decarboxylase (EC 4.1.1.81) and L-Thr kinase activities (EC 2.7.1.177). In Salmonella enterica, where Cba biosynthesis has been extensively studied, the above-mentioned activities are encoded by separate genes, namely, cobD and pduX, respectively. The activities associated with the MM2060 protein (MmCobD) were validated in vitro and in vivo. In vitro, MmCobD used ATP and L-Thr as substrates and generated ADP, L-Thr-P, and (R)-1-aminopropan-2-ol O-phosphate as products. Notably, MmCobD has a 111–amino acid C-terminal extension of unknown function, which contains a putative metal-binding motif. This C-terminal domain alone did not display either activity in vivo or in vitro. Although the C-terminal MmCobD domain was not required for L-Thr-P decarboxylase or L-Thr kinase activities in vivo, its absence negatively affected both activities. In vitro results suggested that this domain may have a regulatory or substrate-gating role. When purified under anoxic conditions, MmCobD displayed Michaelis-Menten kinetics and had 1000-fold higher affinity for ATP and 1300-fold higher catalytic efficiency than MmCobD purified under oxic conditions. To our knowledge, MmCobD is the first example of a new class of L-Thr-P decarboxylases that also have L-Thr kinase activity. An archaeal protein with L-Thr kinase activity had not been identified prior to this work.

SummaryBacteria and archaea use distinct pathways for salvaging exogenous cobinamide (Cbi), a precursor of adenosylcobalamin (coenzyme B12). The bacterial pathway depends on a bifunctional enzyme with kinase and guanylyltransferase activities (CobP in aerobic adenosylcobalamin synthesizers) to convert adenosylcobinamide (AdoCbi) to AdoCbi-guanosine diphosphate (AdoCbi-GDP) via an AdoCbi-phosphate intermediate. Archaea lack CobP, and use a different strategy for the synthesis of AdoCbi-GDP. Archaea cleave off the aminopropanol group of AdoCbi using the CbiZ AdoCbi amidohydrolase to generate adenosylcobyric acid, which is converted to AdoCbiphosphate by the CbiB synthetase, and to AdoCbi-GDP by the CobY guanylyltransferase. We report phylogenetic, in vivo and in vitro evidence that the genome of Rhodobacter sphaeroides encodes functional enzymes for Cbi salvaging systems of both bacterial and archaeal origins. Products of the reactions were identified by high-performance liquid chromatography, UV-visible spectroscopy and bioassay. The cbiZ genes of several bacteria and archaea restored Cbi salvaging in a strain of Salmonella enterica unable to salvage Cbi. Phylogenetic data led us to conclude that CbiZ is an enzyme of archaeal origin that was horizontally transferred to bacteria. Reasons why some bacteria may contain both types of Cbi salvaging systems are discussed.

Several of the enzymes involved in the conversion of adenosylcobyric acid (AdoCby) to adenosylcobamide (AdoCba) are yet to be identified and characterized in some cobamide (Cba)-producing prokaryotes. Using a bioinformatics approach, we identified the bluE gene (locus tag RSP_0788) of Rhodobacter sphaeroides 2.4.1 as a putative functional homolog of the L-threonine kinase enzyme (PduX, EC 2.7.1.177) of S. enterica. In AdoCba, (R)-1-aminopropan-2-ol O-phosphate (AP-P) links the nucleotide loop to the corrin ring; most known AdoCba producers derive AP-P from L-Thr-O-3-phosphate (L-Thr-P). Here, we show that RsBluE has L-Thr-independent ATPase activity in vivo and in vitro. We used P-NMR spectroscopy to show that RsBluE generates L-Thr-P at the expense of ATP and is unable to use L-Ser as a substrate. BluE from R. sphaeroides or Rhodobacter capsulatus restored AdoCba biosynthesis in S. enterica ΕpduX and R. sphaeroides ΕbluE mutant strains. R. sphaeroides ΕbluE strains exhibited a decreased pigment phenotype that was restored by complementation with BluE. Finally, phylogenetic analyses revealed that bluE was restricted to the genomes of a few Rhodobacterales that appear to have a preference for a specific form of Cba, namely Coᴽ-(ᴽ-5,6-dimethylbenzimidazolyl-Coᵦ-adenosylcobamide (a.k.a. adenosylcobalamin, AdoCbl; coenzyme B , CoB ).

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.

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