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Investigating the role of zinc and copper binding motifs of trafficking sites in the cyanobacterium Synechocystis PCC 6803.

ABSTRACT: Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial ( Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaAN and PacSN, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to PacS, but differ in the residues found in their M/IXCXXC metal-binding motifs. To investigate the role of the nonconserved residues in this region on metal binding, the sequence from ZiaAN has been introduced into Atx1 and PacSN, and the motifs of Atx1 and PacSN swapped. The motif sequence can tune Cu(I) affinity only approximately 3-fold. However, the introduction of the ZiaAN motif (MDCTSC) dramatically increases the Zn(II) affinity of both Atx1 and PacSN by up to 2 orders of magnitude. The Atx1 mutant with the ZiaAN motif crystallizes as a side-to-side homodimer very similar to that found for [Cu(I)2-Atx1]2 ( Badarau et al. Biochemistry 2010 , 49 , 7798 ). In a crystal structure of the PacSN mutant possessing the ZiaAN motif (PacSN(ZiaAN)), the Asp residue from the metal-binding motif coordinates Zn(II). This demonstrates that the increased Zn(II) affinity of this variant and the high Zn(II) affinity of ZiaAN are due to the ability of the carboxylate to ligate this metal ion. Comparison of the Zn(II) sites in PacSN(ZiaAN) structures provides additional insight into Zn(II) trafficking in cyanobacteria.

SUBMITTER: Badarau A

PROVIDER: S-EPMC3793899 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Investigating the role of zinc and copper binding motifs of trafficking sites in the cyanobacterium Synechocystis PCC 6803.

Badarau Adriana A Baslé Arnaud A Firbank Susan J SJ Dennison Christopher C

Biochemistry 20130919 39

Although zinc and copper are required by proteins with very different functions, these metals can be delivered to cellular locations by homologous metal transporters within the same organism, as demonstrated by the cyanobacterial ( Synechocystis PCC 6803) zinc exporter ZiaA and thylakoidal copper importer PacS. The N-terminal metal-binding domains of these transporters (ZiaAN and PacSN, respectively) have related ferredoxin folds also found in the metallochaperone Atx1, which delivers copper to ...[more]

PMID: 24050657

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Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Dataset Information

Investigating the role of zinc and copper binding motifs of trafficking sites in the cyanobacterium Synechocystis PCC 6803.

Publications

Investigating the role of zinc and copper binding motifs of trafficking sites in the cyanobacterium Synechocystis PCC 6803.

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