In Vivo Analysis of Cobinamide Salvaging in Rhodobacter sphaeroides Strain 2.4.1

The genome of Rhodobacter sphaeroides encodes the components of two distinct pathways for salvaging cobinamide (Cbi), a precursor of adenosylcobalamin (AdoCbl, coenzyme B₁₂). One pathway, conserved among bacteria, depends on a bifunctional kinase/guanylyltransferase (CobP) enzyme to convert adenosylcobinamide (AdoCbi) to AdoCbi-phosphate (AdoCbi-P), an intermediate in de novo AdoCbl biosynthesis. The other pathway, of archaeal origin, depends on an AdoCbi amidohydrolase (CbiZ) enzyme to generate adenosylcobyric acid (AdoCby), which is converted to AdoCbi-P by the AdoCbi-P synthetase (CobD) enzyme. Here we report that R. sphaeroides strain 2.4.1 synthesizes AdoCbl de novo and that it salvages Cbi using both of the predicted Cbi salvaging pathways. AdoCbl produced by R. sphaeroides was identified and quantified by high-performance liquid chromatography and bioassay. The deletion of cobB (encoding an essential enzyme of the de novo corrin ring biosynthetic pathway) resulted in a strain of R. sphaeroides that would not grow on acetate in the absence of exogenous corrinoids. The results from a nutritional analysis showed that the presence of either CbiZ or CobP was necessary and sufficient for Cbi salvaging, that CbiZ-dependent Cbi salvaging depended on the presence of CobD, and that CobP-dependent Cbi salvaging occurred in a cbiZ⁺ strain. Possible reasons why R. sphaeroides maintains two distinct pathways for Cbi salvaging are discussed.Cobamides, such as adenosylcobalamin (AdoCbl, coenzyme B₁₂), are a group of complex cobalt-containing cyclic tetrapyrrole cofactors whose biosynthesis by bacteria and archaea requires substantial genetic information (>25 genes) (reviewed in references 25, 47, and 56). Two pathways for the de novo synthesis of the corrin ring have been described on the basis of the timing of cobalt insertion into the ring. The late cobalt insertion or aerobic pathway has been well studied in Pseudomonas denitrificans (9), while the early cobalt insertion or anaerobic pathway has been best studied in Salmonella enterica serovar Typhimurium LT2 (25). Many organisms, including those that synthesize AdoCbl de novo, salvage incomplete corrinoids (e.g., cobinamide [Cbi]) from their environments and use them as precursors for the synthesis of complete cobamide cofactors. Cbi is not an intermediate of the de novo AdoCbl biosynthesis pathway but can be converted into one by a process known as Cbi salvaging (Fig. ​(Fig.1)1) (24).Open in a separate windowFIG. 1.Abbreviated view of cobinamide salvaging pathways. Corrin ring-containing intermediates are in bold text. The letter A indicates the de novo corrin ring biosynthesis pathway. Abbreviations: Ado-, adenosyl-; AP, 1-amino-2-propanol; AP-P, 1-amino-2-propanol-phosphate; CobB, hydrogenobyrinic acid a,c-diamide synthase; CobD, adenosylcobinamide-phosphate synthetase; CobP, NTP:adenosylcobinamide kinase, GTP:adenosylcobinamide-phosphate guanylyltransferase; CobY, GTP:adenosylcobinamide-phosphate guanylyltransferase; CbiZ, adenosylcobinamide amidohydrolase. Functional groups are indicated as follows: Me, methyl; Ac, acetamide; and Pr, propionamide.The first step of Cbi salvaging is adenosylation of the molecule to adenosylcobinamide (AdoCbi) (24). The adenosyltransferases which catalyze this reaction are broadly distributed throughout the three domains of life (13, 14, 20, 32, 38). Two distinct pathways for converting AdoCbi into an intermediate of the de novo AdoCbl biosynthesis pathway have been described for prokaryotes. One, which is to date found only in bacteria, relies on a bifunctional nucleoside triphosphate (NTP):AdoCbi kinase (EC 2.7.7.62), GTP:AdoCbi-phosphate (AdoCbi-P) guanylyltransferase (EC 2.7.1.156) enzyme (called CobP in P. denitrificans and CobU in S. Typhimurium), which phosphorylates AdoCbi to AdoCbi-P and converts AdoCbi-P to AdoCbi-GDP (10, 41, 55).Previous work from our laboratory has shown that archaea lack the bifunctional NTP:AdoCbi kinase, GTP:AdoCbi-P guanylyltransferase enzyme and rely on a second pathway for Cbi salvaging (54, 62). In this pathway, AdoCbi is converted to adenosylcobyric acid (AdoCby) by an AdoCbi amidohydrolase (EC 3.5.1.90) known as CbiZ (58, 59, 62). The conversion of AdoCbi-P to AdoCbi-GDP for de novo AdoCbl biosynthesis in archaea is catalyzed by a monofunctional GTP:AdoCbi-P guanylyltransferase (EC 2.7.7.62) called CobY (54, 60), which has not been found in any bacterium.We recently showed that a small percentage of bacterial genomes encode orthologs of both CobP-type and CbiZ-type Cbi salvaging enzymes, raising the question of why these organisms might contain two redundant Cbi salvaging systems (29). A phylogenetic analysis showed that CbiZ has its roots in the archaea and that the cbiZ gene was acquired by several bacterial lineages via horizontal gene transfer.We previously showed that the CbiZ and CobP enzymes from the photosynthetic alphaproteobacterium Rhodobacter sphaeroides are functional in vitro and in vivo in a heterologous complementation system (29). However, the question of how the two Cbi salvaging systems might function in R. sphaeroides remained unresolved.In this paper, we show that R. sphaeroides 2.4.1 synthesizes substantial amounts of cobalamin (Cbl) and that it salvages incomplete corrinoids from its environment. We present in vivo genetic evidence that both the bacterial-type CobP-dependent and archaeal-type CbiZ-dependent Cbi salvaging pathways are functional in this organism. This work represents the first in vivo genetic analysis of coenzyme B₁₂ synthesis and salvaging in R. sphaeroides.