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Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly(cis-1,4-isoprene) degradation.

ABSTRACT: Gordonia westfalica Kb1 and Gordonia polyisoprenivorans VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in G. polyisoprenivorans VH2, the sodA gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(cis-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiments revealed that SodA activity is required for optimal growth on poly(cis-1,4-isoprene), whereas this enzyme has no effect on aerobic growth in the presence of water-soluble substrates like succinate, acetate, and propionate. This was detected by activity staining, and proof of expression was by antibody detection of SOD. When SodA from G. westfalica Kb1 was heterologously expressed in the sodA sodB double mutant Escherichia coli QC779, the recombinant mutant exhibited increased resistance to paraquat, thereby indicating the functionality of the G. westfalica Kb1 SodA and indirectly protection of G. westfalica cells by SodA from oxidative damage. Both sodA from G. polyisoprenivorans VH2 and sodA from G. westfalica Kb1 coded for polypeptides comprising 209 amino acids and having approximately 90% and 70% identical amino acids, respectively, to the SodA from Mycobacterium smegmatis strain MC(2) 155 and Micrococcus luteus NCTC 2665. As revealed by activity staining experiments with the wild type and the disruption mutant of G. polyisoprenivorans, this bacterium harbors only one active SOD belonging to the manganese family. The N-terminal sequences of the extracellular SodA proteins of both Gordonia species showed no evidence of leader peptides for the mature proteins, like the intracellular SodA protein of G. polyisoprenivorans VH2, which was purified under native conditions from the cells. In G. westfalica Kb1 and G. polyisoprenivorans VH2, SodA probably provides protection against reactive oxygen intermediates which occur during degradation of poly(cis-1,4-isoprene).

SUBMITTER: Schulte C

PROVIDER: S-EPMC2607186 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly(cis-1,4-isoprene) degradation.

Schulte Carina C Arenskötter Matthias M Berekaa Mahmoud M MM Arenskötter Quyen Q Priefert Horst H Steinbüchel Alexander A

Applied and environmental microbiology 20081024 24

Gordonia westfalica Kb1 and Gordonia polyisoprenivorans VH2 induce the formation of an extracellular superoxide dismutase (SOD) during poly(cis-1,4-isoprene) degradation. To investigate the function of this enzyme in G. polyisoprenivorans VH2, the sodA gene was disrupted. The mutants exhibited reduced growth in liquid mineral salt media containing poly(cis-1,4-isoprene) as the sole carbon and energy source, and no SOD activity was detectable in the supernatants of the cultures. Growth experiment ...[more]

PMID: 18952871

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Project description:The free radical scavenger WR1065 (SH) is the active thiol form of the clinically approved cytoprotector amifostine. At doses of 40 microM and 4 mM it can activate the redox-sensitive nuclear transcription factor kappaB (NFkappaB) and elevate the expression of the antioxidant gene manganese superoxide dismutase (MnSOD) in human microvascular endothelial cells (HMEC). MnSOD contains binding motifs for a number of transcription factors other than NFkappaB and codes for a potent antioxidant enzyme localized in the mitochondria that is known to confer enhanced radiation resistance to cells. The purpose of this study was to determine the effect of WR1065 exposure on the various transcription factors known to affect MnSOD expression along with the subsequent kinetics of intracellular elevation of MnSOD protein levels and associated change in sensitivity to ionizing radiation in HMEC. Cells were grown to confluence and exposed to WR1065 for 30 min. Affects on the transcription factors AP1, AP2, CREB, NFkappaB, and Sp1 were monitored as a function of time ranging from 30 min to 4 h after drug exposure using a gel-shift assay. Only NFkappaB exhibited a marked activation and that occurred 30 min following the cessation of drug exposure. MnSOD protein levels, as determined by Western blot analysis, increased up to 16-fold over background control levels by 16 h following drug treatment, and remained at 10-fold or higher levels for an additional 32 h. MnSOD activity was evaluated using a gel-based assay and was found to be active throughout this time period. HMEC were irradiated with X-rays either in the presence of 40 microM or 4 mM WR1065 or 24 h after its removal when MnSOD levels were most elevated. No protection was observed for cells irradiated in the presence of 40 microM WR1065. In contrast, a 4 mM dose of WR1065 afforded an increase in cell survival by a factor of 2. A "delayed radioprotective" effect was, however, observed when cells were irradiated 24 h later, regardless of the concentration of WR1065 used. This effect is characterized as an increase in survival at the 2 Gy dose point, i.e., a 40% increase in survival, and an increase in the initial slope of the survival curve by a factor of 2. The anti-inflammatory sesquiterpene lactone, Helenalin, is an effective inhibitor of NFkappaB activation. HMEC were exposed to Helenalin for 2 h at a nontoxic concentration of 5 microM prior to exposure to WR1065. This treatment not only inhibited activation of NFkappaB by WR1065, but also inhibited the subsequent elevation of MnSOD and the delayed radioprotective effect. A persistent marked elevation of MnSOD in cells following their exposure to a thiol-containing reducing agent such as WR1065 can result in an elevated resistance to the cytotoxic effects of ionizing radiation and represents a novel radioprotection paradigm.

Dataset Information

Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly(cis-1,4-isoprene) degradation.

Publications

Possible involvement of an extracellular superoxide dismutase (SodA) as a radical scavenger in poly(cis-1,4-isoprene) degradation.

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