Dataset Information

Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

ABSTRACT: Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammalian cells by expressing bacterial and plant Fds with ferredoxin-NADP+ reductases (FNR). We delineated the rate limiting factors and found that the main metabolic precursor, heme, was not the primary limiting factor for producing either the cyanobacterial or plant chromophores, phycocyanobilin or phytochromobilin, respectively. In fact, Fd is limiting, followed by Fd+FNR and finally heme. Using these findings, we optimized the PCB production system and combined it with a tissue penetrating red/far-red sensing PhyB optogenetic gene switch in animal cells. We further characterized this system in several mammalian cell lines using red and far-red light. Importantly, we found that the light-switchable gene system remains active for several hours upon illumination, even with a short light pulse, and requires very small amounts of light for maximal activation. Boosting chromophore production by matching metabolic pathways with specific ferredoxin systems will enable the unparalleled use of the many PhyB optogenetic tools and has broader implications for optimizing synthetic metabolic pathways.

SUBMITTER: Kyriakakis P

PROVIDER: S-EPMC5820651 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP<sup>+</sup> Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

Kyriakakis Phillip P Catanho Marianne M Hoffner Nicole N Thavarajah Walter W Hu Vincent J VJ Chao Syh-Shiuan SS Hsu Athena A Pham Vivian V Naghavian Ladan L Dozier Lara E LE Patrick Gentry N GN Coleman Todd P TP

ACS synthetic biology 20180124 2

Transplanting metabolic reactions from one species into another has many uses as a research tool with applications ranging from optogenetics to crop production. Ferredoxin (Fd), the enzyme that most often supplies electrons to these reactions, is often overlooked when transplanting enzymes from one species to another because most cells already contain endogenous Fd. However, we have shown that the production of chromophores used in Phytochrome B (PhyB) optogenetics is greatly enhanced in mammali ...[more]

PMID: 29301067

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Project description:Ferredoxin: NADP+ reductase (FNR) is an FAD-containing enzyme best known for catalysing the transfer of electrons from ferredoxin (Fd) to NADP+ to make NADPH during photosynthesis. It is also the prototype for a broad enzyme superfamily, including the NADPH oxidases (NOXs) that all catalyse similar FAD-enabled electron transfers between NAD(P)H and one-electron carriers. Here, we define further mechanistic details of the NAD(P)H ? FAD hydride-transfer step of the reaction based on spectroscopic studies and high-resolution (~ 1.5 Å) crystallographic views of the nicotinamide-flavin interaction in crystals of corn root FNR Tyr316Ser and Tyr316Ala variants soaked with either nicotinamide, NADP+ , or NADPH. The spectra obtained from FNR crystal complexes match those seen in solution and the complexes reveal active site packing interactions and patterns of covalent distortion of the FAD that imply significant active site compression that would favour catalysis. Furthermore, anisotropic B-factors show that the mobility of the C4 atom of the nicotinamide in the FNR:NADP+ complex has a directionality matching that expected for boat-like excursions of the nicotinamide ring thought to enhance hydride transfer. Arguments are made for the relevance of this binding mode to catalysis, and specific consideration is given to how the results extrapolate to provide insight to structure-function relations for the membrane-bound NOX enzymes for which little structural information has been available. DATABASES:Structural data are available in the PDB database under the accession numbers 3LO8 (wild-type), 5VW4 [Y316S:nicotinamide (P32 21)], 5VW9 [Y316S:nicotinamide (P31 21)], 5VW3 [Y316S:NADP+ (P32 21)], 5VW8 [Y316S:NADP+ (P31 21)], 5VW2 [Y316S:NADPH (P32 21)], 5VW5 [Y316A:nicotinamide (P32 21)], 5VW6 [Y316A:NADP+ (P32 21)], 5VW7 [Y316A:NADPH (P32 21)], 5VWA [Y316F (P32 21)], and 5VWB [Y316F:NADP+ (P31 21)]. Enzyme Commission number: ferredoxin:NADP+ reductase - E C1.18.1.2.

Project description:The ferredoxin nicotinamide adenine dinucleotide phosphate reductase from Pseudomonas aeruginosa ( pa-FPR) in complex with NADP (+) has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The structure of the complex revealed that pa-FPR harbors a preformed NADP (+) binding pocket where the cofactor binds with minimal structural perturbation of the enzyme. These findings were complemented by obtaining sequential backbone resonance assignments of this 29518 kDa enzyme, which enabled the study of the pa-FPR-NADP complex by monitoring chemical shift perturbations induced by addition of NADP (+) or the inhibitor adenine dinucleotide phosphate (ADP) to pa-FPR. The results are consistent with a preformed NADP (+) binding site and also demonstrate that the pa-FPR-NADP complex is largely stabilized by interactions between the protein and the 2'-P AMP portion of the cofactor. Analysis of the crystal structure also shows a vast network of interactions between the two cofactors, FAD and NADP (+), and the characteristic AFVEK (258) C'-terminal extension that is typical of bacterial FPRs but is absent in their plastidic ferredoxin NADP (+) reductase (FNR) counterparts. The conformations of NADP (+) and FAD in pa-FPR place their respective nicotinamide and isoalloxazine rings 15 A apart and separated by residues in the C'-terminal extension. The network of interactions among NADP (+), FAD, and residues in the C'-terminal extension indicate that the gross conformational rearrangement that would be necessary to place the nicotinamide and isoalloxazine rings parallel and adjacent to one another for direct hydride transfer between NADPH and FAD in pa-FPR is highly unlikely. This conclusion is supported by observations made in the NMR spectra of pa-FPR and the pa-FPR-NADP complex, which strongly suggest that residues in the C'-terminal sequence do not undergo conformational exchange in the presence or absence of NADP (+). These findings are discussed in the context of a possible stepwise electron-proton-electron transfer of hydride in the oxidation of NADPH by FPR enzymes.

Dataset Information

Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP+ Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

Publications

Biosynthesis of Orthogonal Molecules Using Ferredoxin and Ferredoxin-NADP<sup>+</sup> Reductase Systems Enables Genetically Encoded PhyB Optogenetics.

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