{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Wang PH"],"funding":["Ministry of Science and Technology","National Institutes of Natural Sciences","National Human Genome Research Institute"],"pagination":["2887-2896"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC10594875"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["12(10)"],"pubmed_abstract":["Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O<sub>2</sub> lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system <i>in vitro via</i> a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from <i>E. coli</i>. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O<sub>2</sub>-depleted conditions. To overcome the O<sub>2</sub> lability of [4Fe-4S] Fe-S clusters, an O<sub>2</sub>-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH<sub>2</sub> regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O<sub>2</sub> from the reaction while supplying FADH<sub>2</sub> to the SufBC<sub>2</sub>D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis."],"journal":["ACS synthetic biology"],"pubmed_title":["One-Pot <i>De Novo</i> Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions."],"pmcid":["PMC10594875"],"funding_grant_id":["AB311001","AB301003","111-2628-E-008-009"],"pubmed_authors":["Wang PH","Nishikawa S","McGlynn SE","Fujishima K"],"additional_accession":[]},"is_claimable":false,"name":"One-Pot <i>De Novo</i> Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions.","description":"Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O<sub>2</sub> lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system <i>in vitro via</i> a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from <i>E. coli</i>. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O<sub>2</sub>-depleted conditions. To overcome the O<sub>2</sub> lability of [4Fe-4S] Fe-S clusters, an O<sub>2</sub>-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH<sub>2</sub> regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O<sub>2</sub> from the reaction while supplying FADH<sub>2</sub> to the SufBC<sub>2</sub>D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.","dates":{"release":"2023-01-01T00:00:00Z","publication":"2023 Oct","modification":"2026-06-25T03:22:48.575Z","creation":"2026-06-25T03:08:30.885Z"},"accession":"S-EPMC10594875","cross_references":{"pubmed":["37467114"],"doi":["10.1021/acssynbio.3c00155"]}}