|
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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