Biosynthesis and utilization of structurally diverse norcobamide cofactors in the tetrachloroethene-respiring bacterium Sulfurospirillum multivorans

Keller, Sebastian GND

The bacterium Sulfurospirillum multivorans synthesizes the unusual cobamide norpseudo-B12 (Np-B12) as cofactor of the tetrachloroethene reductive dehalogenase PceA de novo. Np-B12 harbors an adenine as lower base and a unique ethanolamine O-phosphate (EA-P) as linker moiety (nor linker) in its nucleotide loop. Other microbial produced cobamides contain varies lower bases (mainly benzimidazoles (Bza)) and an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker with an additional methyl group compared to EA-P. The nucleotide loop of Np-B12 was previously shown to be important for the binding to PceA. To investigate the norcobamide (NCba) biosynthesis and the specificity of PceA towards Np-B12, S. multivorans was amended with different benzimidazole derivatives during growth. The adenine moiety of Np-B12 was efficiently replaced by each of the tested compounds. An in vitro analysis of the lower ligand activating enzyme SmCobT revealed an unusual regioselectivity towards singly substituted benzimidazoles (i.e. 5-CH3-, 5-OH-, 5-OCH3-Bza). Most Bza-harboring NCbas efficiently replaced Np-B12 in PceA by capturing the same position in the active site. Due to a highly inefficient incorporation into PceA, the formation of 5,6-dimethyl-Bza-NCba had strong negative effects on the PCE metabolism of S. multivorans. The enzyme synthesizing the unique EA-P linker in S. multivorans was identified as novel L-serine O-phosphate (L-Ser-P) decarboxylase SmCobD. Besides L-Ser-P this enzyme additionally decarboxylated L-threonine O-3-phosphate (L-Thr-P) to AP-P in vitro with a significantly reduced conversion rate. At high exogenous concentrations of L-Thr-P S. multivorans predominantly synthesized the AP-P containing cobamide pseudo-B12 that was poorly incorporated into SmPceA affirming its specificity for NCba cofactors. This study investigated the unique cobamide biosynthesis pathway of S. multivorans and discovered an exceptional variability of cobamides producible by the organism.

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Keller, Sebastian: Biosynthesis and utilization of structurally diverse norcobamide cofactors in the tetrachloroethene-respiring bacterium Sulfurospirillum multivorans. Jena 2018.

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