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B6db references: phd.thesis

type Thesis
authors Keller, S.
title Biosynthesis and utilization of structurally diverse norcobamide cofactors in the tetrachloroethene- respiring bacterium Sulfurospirillum multivorans
journal PhD thesis, Friedrich Schiller University Jena
Activity smul.1544
Family smul.1544
sel selected
ui phd_thesis
year (2018)
pages 1-146
abstract The respiratory tetrachloroethene (PCE) reductive dehalogenase PceA of the ε- proteobacterium Sulfurospirillum multivorans harbors the unusual cobamide cofactor norpseudo-B 12 that is de novo synthesized by this bacterium. Norpseudo-B 12 contains an adenine as lower base and a unique ethanolamine O-phosphate (EA-P) as linker moiety in its nucleotide loop. Compared to PceA homologs that utilize cobamides with other bases, mainly benzimidazoles, and an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker with an additional methyl group compared to EA-P, SmPceA displayed a drastically higher activity. The special nucleotide loop of norpseudo-B 12 was previously shown to be involved in binding to PceA. The aims of this study were to investigate the biosynthesis of the linker and lower base of norpseudo-B 12 and to analyze the variability of cobamides producible by S. multivorans plus their effects on the PCE-metabolism. The exogenously applied benzimidazoles (Bza) 5,6- dimethyl-Bza (DMB), 5-methyl-Bza (5-MeBza), Bza, 5-hydroxy-Bza (5-OHBza), and 5-methoxy- Bza (5-OMeBza) efficiently replaced adenine in norpseudo-B 12 . The analysis of the lower ligand activating enzyme SmCobT revealed an activation of the singly substituted 5-OMeBza, 5- OHBza, and 5-MeBza to a mixture of α-ribotides (α-RP) with the substituent at C5 and C6 of the benzimidazole with a predominant synthesis of the C6 isomer only in the case of 5-OHBza activation. Exclusively 5-OMeBza-, 6-OHBza- and both 5- and 6-MeBza norcobamide (NCba) were synthesized by S. multivorans revealing a specificity in the cobamide biosynthesis for certain CobT products. Most (Bza)-NCbas efficiently replaced norpseudo-B 12 in PceA by capturing the same position in the active site, which had no effects on the PCE-metabolism. Only DMB-NCba showed strong negative effects on the PCE-metabolism of S. multivorans due to a highly inefficient incorporation of this cobamide into PceA. The key enzyme synthesizing the unique EA-P linker was analyzed 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 lower conversion rate. At high exogenous concentrations of L- Thr-P S. multivorans predominantly synthesized the AP-P containing pseudo-B 12 , which was poorly incorporated into SmPceA affirming its specificity for NCba cofactors. This study unraveled the special biosynthesis of the unique norpseudo-B 12 in S. multivorans and the exceptional variability of cobamides and norcobamides that can be produced by this organism. Moreover, new relevant insights into the binding of cobamide cofactors to target enzymes were achieved.
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last changed 2019/06/19 10:00

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