The SMUL_1544 Gene Product Governs Norcobamide Biosynthesis in the Tetrachloroethene-Respiring Bacterium Sulfurospirillum multivorans

Abstract

ABSTRACT The tetrachloroethene (PCE)-respiring bacterium Sulfurospirillum multivorans produces a unique cobamide, namely, norpseudo-B 12 , which, in comparison to other cobamides, e.g., cobalamin and pseudo-B 12 , 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 Δ cobD 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-B 12 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-B 12 . 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-B 12 , 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-B 12 linker moiety, which strongly supports the hypothesis of EA-P formation from l -serine- O -phosphate ( l -Ser-P) in this organism. IMPORTANCE The identification of the gene product SMUL_1544 as a putative l -Ser-P decarboxylase involved in norcobamide biosynthesis in S. multivorans adds a novel module to the assembly line of cobamides (complete corrinoids) in prokaryotes. Selected cobamide-containing enzymes (e.g., reductive dehalogenases) showed specificity for their cobamide cofactors. It has recently been proposed that the structure of the linker moiety of norpseudo-B 12 and the mode of binding of the EA-P linker to the PceA enzyme reflect the high specificity of the enzyme for its cofactor. Data reported herein do not support this idea. In fact, norpseudo-B 12 was functional in the cobamide-dependent methionine biosynthesis of S. enterica , raising questions about the role of norcobamides in nature.