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Abstract

The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B12, which, in comparison to other cobamides, e.g., cobalamin and pseudo-B12, lacks the methyl group in the linker moiety of the nucleotide loop. In this study, the protein SMUL_1544 was shown to be responsible for the formation of the unusual linker moiety, which is most probably derived from ethanolamine-phosphate (EA-P) as the precursor. The product of the SMUL_1544 gene successfully complemented a Salmonella enterica ΔcobD mutant. The cobD gene encodes an l-threonine-O-3-phosphate (l-Thr-P) decarboxylase responsible for the synthesis of (R)-1-aminopropan-2-ol O-2-phosphate (AP-P), required specifically for cobamide biosynthesis. When SMUL_1544 was produced in the heterologous host lacking CobD, norpseudo-B12 was formed, which pointed toward the formation of EA-P rather than AP-P. Guided cobamide biosynthesis experiments with minimal medium supplemented with l-Thr-P supported cobamide biosynthesis in S. enterica producing SMUL_1544 or S. multivorans Under these conditions, both microorganisms synthesized pseudo-B12 This observation indicated a flexibility in the SMUL_1544 substrate spectrum. From the formation of catalytically active PCE reductive dehalogenase (PceA) in S. multivorans cells producing pseudo-B12, a compatibility of the respiratory enzyme with the cofactor was deduced. This result might indicate a structural flexibility of PceA in cobamide binding. Feeding of l-[3-(13)C]serine to cultures of S. multivorans resulted in isotope labeling of the norpseudo-B12 linker moiety, which strongly supports the hypothesis of EA-P formation from l-serine-O-phosphate (l-Ser-P) in this organism.