Dataset Information

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

ABSTRACT: The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the -10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.

SUBMITTER: Bender KS

PROVIDER: S-EPMC126438 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature

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Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

Bender Kelly S KS O'Connor Susan M SM Chakraborty Romy R Coates John D JD Achenbach Laurie A LA

Applied and environmental microbiology 20021001 10

The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence iden ...[more]

PMID: 12324326

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Project description:The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2 -) to yield Cl- and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2 -) with the evolution of O2 (k cat = (2.0±0.2)×102 s-1; k cat/K M = (1.2±0.2)×105 M-1 s-1 at pH 5.2). Stopped-flow studies of this BrO2 - decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK a of 6.7±0.1 in good agreement with the pK a of DaCld activity with ClO2 -. The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having pK a 6.7. Examination of reaction mixtures comprising DaCld and ClO2 - by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO2, which forms in preference to the analogous to AA• in the BrO2 - reaction. Addition of ClO- to Cpd II did not yield O2. Together these results are consistent with heterolytic cleavage of the O-BrO- and O-ClO- bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

Dataset Information

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

Publications

Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe.

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