Dataset Information

Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1.

ABSTRACT: Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX(2)CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escherichia coli as host organism. Several reporter c-type cytochromes were employed including cytochrome c nitrite reductases (NrfA) from E. coli and Campylobacter jejuni that differ in their active-site HBMs (CX(2)CK or CX(2)CH). W. succinogenes CcsA2 was found to attach haem to standard CX(2)CH motifs in various cytochromes whereas other HBMs were not recognized. NrfI was able to attach haem c to the active-site CX(2)CK motif of both W. succinogenes and E. coli NrfA, but not to NrfA from C. jejuni. Different apo-cytochrome variants carrying the CX(15)CH motif, assumed to be recognized by CcsA1 during maturation of the octahaem cytochrome MccA, were not processed by CcsA1 in either W. succinogenes or E. coli. It is concluded that the dedicated CCHLs NrfI and CcsA1 attach haem to non-standard HBMs only in the presence of further, as yet uncharacterized structural features. Interestingly, it proved impossible to delete the ccsA2 gene from the W. succinogenes genome, a finding that is discussed in the light of the available genomic, proteomic and functional data on W. succinogenes c-type cytochromes.

SUBMITTER: Kern M

PROVIDER: S-EPMC3414424 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1.

Kern Melanie M Eisel Florian F Scheithauer Juliane J Kranz Robert G RG Simon Jörg J

Molecular microbiology 20091117 1

Bacterial c-type cytochrome maturation is dependent on a complex enzymic machinery. The key reaction is catalysed by cytochrome c haem lyase (CCHL) that usually forms two thioether bonds to attach haem b to the cysteine residues of a haem c binding motif (HBM) which is, in most cases, a CX(2)CH sequence. Here, the HBM specificity of three distinct CCHL isoenzymes (NrfI, CcsA1 and CcsA2) from the Epsilonproteobacterium Wolinella succinogenes was investigated using either W. succinogenes or Escher ...[more]

PMID: 19919672

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Project description:C(4)-dicarboxylate transport is a prerequisite for anaerobic respiration with fumarate in Wolinella succinogenes, since the substrate site of fumarate reductase is oriented towards the cytoplasmic side of the membrane. W. succinogenes was found to transport C(4)-dicarboxylates (fumarate, succinate, malate, and aspartate) across the cytoplasmic membrane by antiport and uniport mechanisms. The electrogenic uniport resulted in dicarboxylate accumulation driven by anaerobic respiration. The molar ratio of internal to external dicarboxylate concentration was up to 10(3). The dicarboxylate antiport was either electrogenic or electroneutral. The electroneutral antiport required the presence of internal Na(+), whereas the electrogenic antiport also operated in the absence of Na(+). In the absence of Na(+), no electrochemical proton potential (delta p) was measured across the membrane of cells catalyzing fumarate respiration. This suggests that the proton potential generated by fumarate respiration is dissipated by the concomitant electrogenic dicarboxylate antiport. Three gene loci (dcuA, dcuB, and dctPQM) encoding putative C(4)-dicarboxylate transporters were identified on the genome of W. succinogenes. The predicted gene products of dcuA and dcuB are similar to the Dcu transporters that are involved in the fumarate respiration of Escherichia coli with external C(4)-dicarboxylates. The genes dctP, -Q, and -M probably encode a binding-protein-dependent secondary uptake transporter for dicarboxylates. A mutant (DcuA(-) DcuB(-)) of W. succinogenes lacking the intact dcuA and dcuB genes grew by nitrate respiration with succinate as the carbon source but did not grow by fumarate respiration with fumarate, malate, or aspartate as substrates. The DcuA(-), DcuB(-), and DctQM(-) mutants grew by fumarate respiration as well as by nitrate respiration with succinate as the carbon source. Cells of the DcuA(-) DcuB(-) mutant performed fumarate respiration without generating a proton potential even in the presence of Na(+). This explains why the DcuA(-) DcuB(-) mutant does not grow by fumarate respiration. Growth by fumarate respiration appears to depend on the function of the Na(+)-dependent, electroneutral dicarboxylate antiport which is catalyzed exclusively by the Dcu transporters. Dicarboxylate transport via the electrogenic uniport is probably catalyzed by the DctPQM transporter and by a fourth, unknown transporter that may also operate as an electrogenic antiporter.

Dataset Information

Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1.

Publications

Substrate specificity of three cytochrome c haem lyase isoenzymes from Wolinella succinogenes: unconventional haem c binding motifs are not sufficient for haem c attachment by NrfI and CcsA1.

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