Dataset Information

Structural and Functional Characterization of CreFH1, the Frataxin Homolog from Chlamydomonas reinhardtii.

ABSTRACT: Frataxin plays a key role in cellular iron homeostasis of different organisms. It has been implicated in iron storage, detoxification, delivery for Fe-S cluster assembly and heme biosynthesis. However, its specific role in iron metabolism remains unclear, especially in photosynthetic organisms. To gain insight into the role and properties of frataxin in algae, we identified the gene CreFH1, which codes for the frataxin homolog from Chlamydomonas reinhardtii. We performed the cloning, expression and biochemical characterization of CreFH1. This protein has a predicted mitochondrial transit peptide and a significant structural similarity to other members of the frataxin family. In addition, CreFH1 was able to form a dimer in vitro, and this effect was increased by the addition of Cu²⁺ and also attenuated the Fenton reaction in the presence of a mixture of Fe²⁺ and H₂O₂. Bacterial cells with overexpression of CreFH1 showed increased growth in the presence of different metals, such as Fe, Cu, Zn and Ni and H₂O₂. Thus, results indicated that CreFH1 is a functional protein that shows some distinctive features compared to its more well-known counterparts, and would play an important role in response to oxidative stress in C. reinhardtii.

SUBMITTER: Terenzi A

PROVIDER: S-EPMC9331050 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Structural and Functional Characterization of CreFH1, the Frataxin Homolog from <i>Chlamydomonas reinhardtii</i>.

Terenzi Agustina A Pagani Maria A MA Gomez-Casati Diego F DF Busi Maria V MV

Plants (Basel, Switzerland) 20220726 15

Frataxin plays a key role in cellular iron homeostasis of different organisms. It has been implicated in iron storage, detoxification, delivery for Fe-S cluster assembly and heme biosynthesis. However, its specific role in iron metabolism remains unclear, especially in photosynthetic organisms. To gain insight into the role and properties of frataxin in algae, we identified the gene CreFH1, which codes for the frataxin homolog from <i>Chlamydomonas reinhardtii</i>. We performed the cloning, expr ...[more]

PMID: 35893635

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Project description:BackgroundRecent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the C. reinhardtii genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI), prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale.ResultsWe assigned enzymatic functions to the translated JGI ORF models of C. reinhardtii by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST. In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate.ConclusionsWe functionally annotated approximately 1,400 JGI predicted metabolic ORFs that can facilitate the reconstruction and refinement of a genome-scale metabolic network. The unveiling of the metabolic potential of this organism, along with structural verification of the relevant ORFs, facilitates the selection of metabolic engineering targets with applications in bioenergy and biopharmaceuticals. The ORF clones are a resource for downstream studies.

Dataset Information

Structural and Functional Characterization of CreFH1, the Frataxin Homolog from Chlamydomonas reinhardtii.

Publications

Structural and Functional Characterization of CreFH1, the Frataxin Homolog from <i>Chlamydomonas reinhardtii</i>.

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