Cobamide Biosynthesis: Examination of Aminopropanol Production and Incorporation

Amanda Abavana

doi:10.17918/00010604

The gut microbiome is a complex community whose stability requires metabolic interactions between microbes, including cross-feeding of nutrients like cobamides. Cobamides are a family of organometallic co-factors including vitamin B12, which supports metabolism in all three domains of life. They are composed of a central contracted tetrapyrrole ring which chelates a central cobalt ion, an aminopropanol phosphate (APP) arm, and a nucleotide loop containing a variable lower ligand. Humans require the vitamin B12 analog (also referred to as cobalamin), which contains 5,6-dimethylbenzimidazole as the lower ligand. Vitamin B12 is required for DNA synthesis and nerve cell maintenance, and deficiencies can lead to serious health complications like anemia and neurological disorders. Humans must acquire vitamin B12 from dietary sources because cobamides are distributed through food webs but are ultimately only produced by certain prokaryotes. Interestingly, the human gut microbiome is a rich source of diverse cobamides, although vitamin B12 is a minor component and cannot be absorbed across the large intestine. Gut bacteria utilize cobamides to support function of a range of pathways, including methionine production via methionine synthase (MetH), propionic acid production via methylmalonyl-CoA mutase (McmA), and catabolism of food substrates such as ethanolamine and 1,2-propanediol. While most bacteria can utilize cobamides, only some can produce them through a complex pathway involving ~30 enzymatic steps. For APP production in Salmonella enterica, PduX phosphorylates L-threonine (L-Thr) and CobD decarboxylates it, and the resulting APP gets incorporated with the cobyric acid intermediate by CbiB. However, PduX and CobD are poorly conserved among known and predicted de novo cobamide producers, including Pseudomonas species. Here we examine biosynthesis and incorporation of the APP arm in Pseudomonas species using liquid chromatography mass spectrometry. We performed stable isotope studies to verify that L-threonine is a precursor of APP and used heterologous CbiB assays to assess a putative alternate pathway. Our results suggest that Pseudomonas species encode an alternate kinase, and further studies will identify the kinase and determine whether this pathway is shared by other cobamide producers. Improved characterization of cobamide biosynthesis will help define cobamide cross-feeding interactions vital for healthy and stable gut microbiomes.

Cobamide Biosynthesis: Examination of Aminopropanol Production and Incorporation

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Cobamide Biosynthesis: Examination of Aminopropanol Production and Incorporation

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