Exogenous 5,6‐dimethylbenzimidazole caused production of a non‐functional tetrachloroethene reductive dehalogenase in Sulfurospirillum multivorans

Abstract

Corrinoid-dependent reductive dehalogenation is mediated by phylogenetically diverse anaerobic bacteria that either synthesize corrinoids de novo or are dependent on corrinoid salvaging from the environment. The tetrachloroethene (PCE) reductive dehalogenase (PceA) of the Gram-negative Epsilonproteobacterium Sulfurospirillum multivorans harbours a norpseudo-B12 as corrinoid cofactor. Norpseudo-B12 differs from coenzyme B12 in the nucleotide loop structure. Adenine instead of 5,6-dimethylbenzimidazole (DMB) serves as lower ligand base of the central cobalt ion, and the nucleotide loop of norpseudo-B12 lacks a methyl group at position 176. In this study, S. multivorans was grown anaerobically with PCE in the presence of DMB. At a DMB concentration of 25 μM, the adenine moiety in the nucleotide loop of norpseudo-B12 was quantitatively replaced by DMB. The formation of the DMB-containing nor-B12 severely affected PCE-dependent growth and the PceA activity. In DMB-treated cells processing of the cytoplasmic PceA precursor was impeded, a result pointing to retarded cofactor incorporation. PceA enriched from cells cultivated with DMB contained nor-B12 . Nor-B12 purified from cells grown in the presence of DMB mediated the abiotic reductive dehalogenation of trichloroacetate to dichloroacetate at a 25-fold lower rate in comparison with norpseudo-B12 , a fact underpinning the relevance of norpseudo-B12 as efficient catalyst for reductive dehalogenation in general.

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

“…Hence, the PceA structure might adopt a conformation which allows the binding of either norpseudo-B 12 or pseudo-B 12 . Such an acceptance of a structurally different cobamide cofactor by PceA has not been observed before, when the lower base of the nucleotide loop in norpseudo-B 12 was exchanged from adenine to 5,6-dimethylbenzimidazole (23). Current work in our laboratory seeks to determine whether or not catalytically active PceA from cells cultivated in the presence of L-Thr-P contains pseudo-B 12 or whether it selectively binds traces of norpseudo-B 12 .…”

“…For cobamide extraction, S. multivorans was grown on pyruvate-and fumarate-containing medium. The procedure for cobamide extraction and analysis via high-performance liquid chromatography (HPLC) is described elsewhere (23). High-resolution mass spectra were recorded using a Bruker Maxis high-resolution quantitative time of flight mass spectrometer equipped with an electrospray ionization source operated in the positive mode (Bruker Daltonik GmbH, Bremen, Germany).…”

“…HPLC separations were achieved using a Dionex UltiMate 3000 instrument equipped with a Phenomenex Gemini C 18 column (250 by 2 mm, 5 m) operated at a flow rate of 400 l/min starting with 3% aqueous acetonitrile for 5 min, followed by a linear gradient to 100% acetonitrile within 35 min, using 0.5% acetic acid as an additive. PceA activity measurement and immunological detection of the enzyme in crude cell extracts of S. multivorans cells were performed as previously described (23).…”

Functional Heterologous Production of Reductive Dehalogenases from Desulfitobacterium hafniense Strains

“…No negative effect of exogenous DMB on the PCE-dependent growth or dechlorination rate has been observed in D. hafniense Y51 or DCB-2 cells (unpublished results) in contrast to the results seen with the PCE-dechlorinating Sulfurospirillum multivorans. In the latter organism, PCE-dependent growth was impaired by the presence of DMB (33). The presence of DMB in the growth medium of the S. blattae expression strains resulted in a higher intracellular cobamide level accompanied by higher activity of the recombinant RdhA enzyme.…”

“…The purification and HPLC analysis of cobamides were performed as described earlier by Keller et al (33). For the subsequent mass spectrometric analysis, the cobamides were applied to an ultra-high-performance liquid chromatography (UHPLC) system using an Ultimate 3000 series rapid-separation liquid chromatography (RSLC) system (Dionex, Sunnyvale, CA) connected to a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany).…”