<i>Shewanella oneidensis</i> MR-1 is a Gram-negative bacterium isolated from anaerobic freshwater lake sediments of Lake Oneida that exhibits remarkable respiratory versatility. In the absence of molecular oxygen, <i>S. oneidensis</i> MR-1 couples anaerobic growth to the reduction of various substrates, including ferric iron (FeIII), thiosulfate (S<sub>2</sub>O<sub>3</sub><sup>2-</sup>), sulfite (SO<sub>3</sub><sup>2-</sup>), trimethylamine <i>N-</i>oxide (TMAO), nitrate (NO<sub>3</sub><sup>-</sup>), nitrite (NO<sub>2</sub><sup>-</sup>) and organic substrates such was fumarate. The metabolic flexibility of <i>S. oneidensis</i> MR-1 is coupled to a complex and branched anaerobic respiratory chain. The respiratory enzymes of the fumarate reduction pathway have been extensively studied in <i>S. oneidensis</i> MR-1 and the related marine bacterium, <i>S. frigidimarina</i> NCIMB400. The terminal fumarate reductase of <i>Shewanella </i>is a soluble periplasmic flavocytochrome c<sub>3</sub> (Fcc<sub>3</sub>) that catalyses the unidirectional production of succinate. The X-ray crystal structure of Fcc<sub>3</sub> solved to high resolution provided the first detailed insight into the catalytic mechanism of fumarate reduction. In this work, the Fcc<sub>3</sub> X-ray crystal structure provided a structural template to construct homology models of related flavoenzymes of unknown structure and function. The novel flavoenzymes were identified by sequence analysis of the <i>S. oneidensis</i> MR-1 genome and were shown to comprise separately encoded flavin (FccA54, FccA56 and FccA342) and cytochrome subunits (FccB54, FccB56 and FccB342), respectively, that were related by sequence to the corresponding domains in Fcc<sub>3</sub>. Molecular modelling of the catalytic flavin-binding subunits led to the suggestion that these related enzymes catalyse the reduction of acrylate-like substrates. Several biologically relevant plant metabolites, including phenylacrylates incorporated into lignin, were identified as potential substrates of the Fcc<sub>3</sub>-like enzymes. An <i>fccA54</i> and <i>fccB54</i> knockout strain of <i>S. oneidensis</i> MR-1 (MB5415) was constructed and grown anaerobically with each of the candidate acrylates to ascertain the biological function of FccA54.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:641618 |
| Date | January 2003 |
| Creators | Bilsland, Morag |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/10812 |
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