One-Pot Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System Under Aerobic Conditions

Overview

Journal ACS Synth Biol

Publisher American Chemical Society

Specialties Biotechnology
Molecular Biology

Date 2023 Jul 19

PMID 37467114

Authors

Po-Hsiang Wang

Shota Nishikawa

Shawn Erin McGlynn

Kosuke Fujishima

Affiliations

Soon will be listed here.

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 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 a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from . Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O-depleted conditions. To overcome the O lability of [4Fe-4S] Fe-S clusters, an O-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 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O from the reaction while supplying FADH to the SufBCD 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.

Citing Articles

Cofactor recycling strategies for secondary metabolite production in cell-free protein expression systems.

Zou Y, Bailey C Biophys Rev. 2024; 16(5):591-603.

PMID: 39618802 PMC: 11604874. DOI: 10.1007/s12551-024-01234-1.

References

Motohashi K . Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis. Methods Mol Biol. 2016; 1498:349-357. DOI: 10.1007/978-1-4939-6472-7_23. View

Kennedy M, Mende-Mueller L, BLONDIN G, BEINERT H . Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein. Proc Natl Acad Sci U S A. 1992; 89(24):11730-4. PMC: 50630. DOI: 10.1073/pnas.89.24.11730. View

Monteiro T, Moreira M, Gaspar S, Almeida M . Bilirubin oxidase as a single enzymatic oxygen scavenger for the development of reductase-based biosensors in the open air and its application on a nitrite biosensor. Biosens Bioelectron. 2022; 217:114720. DOI: 10.1016/j.bios.2022.114720. View

Boyer M, Stapleton J, Kuchenreuther J, Wang C, Swartz J . Cell-free synthesis and maturation of [FeFe] hydrogenases. Biotechnol Bioeng. 2007; 99(1):59-67. DOI: 10.1002/bit.21511. View

Guth-Metzler R, Bray M, Frenkel-Pinter M, Suttapitugsakul S, Montllor-Albalate C, Bowman J . Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage. Nucleic Acids Res. 2020; 48(15):8663-8674. PMC: 7470983. DOI: 10.1093/nar/gkaa586. View

Johnson M . Iron-sulfur proteins: new roles for old clusters. Curr Opin Chem Biol. 1998; 2(2):173-81. DOI: 10.1016/s1367-5931(98)80058-6. View

Patil P, Ballou D . The use of protocatechuate dioxygenase for maintaining anaerobic conditions in biochemical experiments. Anal Biochem. 2000; 286(2):187-92. DOI: 10.1006/abio.2000.4802. View

Shimizu Y, Inoue A, Tomari Y, Suzuki T, Yokogawa T, Nishikawa K . Cell-free translation reconstituted with purified components. Nat Biotechnol. 2001; 19(8):751-5. DOI: 10.1038/90802. View

Layer G, Gaddam S, Ayala-Castro C, Ollagnier-de Choudens S, Lascoux D, Fontecave M . SufE transfers sulfur from SufS to SufB for iron-sulfur cluster assembly. J Biol Chem. 2007; 282(18):13342-50. DOI: 10.1074/jbc.M608555200. View

10.

Garcia P, DAngelo F, de Choudens S, Dussouchaud M, Bouveret E, Gribaldo S . An early origin of iron-sulfur cluster biosynthesis machineries before Earth oxygenation. Nat Ecol Evol. 2022; 6(10):1564-1572. DOI: 10.1038/s41559-022-01857-1. View

11.

Zheng L, Cash V, Flint D, Dean D . Assembly of iron-sulfur clusters. Identification of an iscSUA-hscBA-fdx gene cluster from Azotobacter vinelandii. J Biol Chem. 1998; 273(21):13264-72. DOI: 10.1074/jbc.273.21.13264. View

12.

Aitken C, Marshall R, Puglisi J . An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J. 2007; 94(5):1826-35. PMC: 2242739. DOI: 10.1529/biophysj.107.117689. View

13.

Beinert H, Kennedy M, Stout C . Aconitase as Ironminus signSulfur Protein, Enzyme, and Iron-Regulatory Protein. Chem Rev. 1996; 96(7):2335-2374. DOI: 10.1021/cr950040z. View

14.

Ragsdale S . Pyruvate ferredoxin oxidoreductase and its radical intermediate. Chem Rev. 2003; 103(6):2333-46. DOI: 10.1021/cr020423e. View

15.

Wang P, Fujishima K, Berhanu S, Kuruma Y, Jia T, Khusnutdinova A . A Bifunctional Polyphosphate Kinase Driving the Regeneration of Nucleoside Triphosphate and Reconstituted Cell-Free Protein Synthesis. ACS Synth Biol. 2019; 9(1):36-42. DOI: 10.1021/acssynbio.9b00456. View

16.

Evans M, Buchanan B, Arnon D . A new ferredoxin-dependent carbon reduction cycle in a photosynthetic bacterium. Proc Natl Acad Sci U S A. 1966; 55(4):928-34. PMC: 224252. DOI: 10.1073/pnas.55.4.928. View

17.

Gaudu P, Touati D, Niviere V, Fontecave M . The NAD(P)H:flavin oxidoreductase from Escherichia coli as a source of superoxide radicals. J Biol Chem. 1994; 269(11):8182-8. View

18.

Tsibris J, Tsai R, GUNSALUS I, Orme-Johnson W, HANSEN R, BEINERT H . The number of iron atoms in the paramagnetic center (G =1.94) of reduced putidaredoxin, a nonheme iron protein. Proc Natl Acad Sci U S A. 1968; 59(3):959-65. PMC: 224789. DOI: 10.1073/pnas.59.3.959. View

19.

Takahashi Y, Tokumoto U . A third bacterial system for the assembly of iron-sulfur clusters with homologs in archaea and plastids. J Biol Chem. 2002; 277(32):28380-3. DOI: 10.1074/jbc.C200365200. View

20.

Chang A, Jeske L, Ulbrich S, Hofmann J, Koblitz J, Schomburg I . BRENDA, the ELIXIR core data resource in 2021: new developments and updates. Nucleic Acids Res. 2020; 49(D1):D498-D508. PMC: 7779020. DOI: 10.1093/nar/gkaa1025. View