Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Kim, Young‐Sug; Hara, Masayuki; Miyake, Jun; Ohkawa, Hideo; Karube, Isao

doi:10.1007/bf00222553

Cited by 14 publications

(10 citation statements)

References 3 publications

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“…Several can be induced by light (37), whereas others are induced by plant stresses such as wounding, pathogens, or xenobiotic compounds (38). Xenobiotic induction of P450s in animal systems (such as birds and fish) has led to its use as an indicator of environmental contamination (39,40).…”

Section: Phytotransformation Enzymology and Biochemistrymentioning

confidence: 99%

Advances in Phytoremediation

Dietz¹,

Schnoor²

2001

Environmental Health Perspectives

100

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Section: Phytotransformation Enzymology and Biochemistrymentioning

confidence: 99%

Advances in Phytoremediation

Dietz¹,

Schnoor²

2001

Environmental Health Perspectives

100

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“…Thus, the midpoint redox potential of cyanobacterial ferredoxins of between −325 and −390 mV, is sufficient to reduce the P450. Indeed, it has been shown with in vitro experiments using chloroplasts or purified thylakoids and spinach ferredoxin that electrons derived from photosystem I (PSI) can be redirected to heterologous P450s to provide reducing equivalents in a light-dependent manner. ,− In these in vitro experiments, ferredoxin serves as the electron donor instead of NADPH.…”

mentioning

confidence: 99%

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

et al. 2016

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Photosynthesis is the pivotal biochemical reaction on the planet, providing energy for the 30 global ecosystem. The evolution of oxygenic photosynthesis 2.7-3.2 billion years ago led to 31 the oxygenation of the planet. 1 This subsequently hampered the efficiency of 32 photosynthesis as oxygen competed for binding to the active site of the carbon-fixing 33 catalyst ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). As a result, the 34 potential of photosynthesis is not achieved as the capacity for light capture and electron 35 transport is often greater than the capacity for carbon-fixation. Photosynthetic cells therefore 36 lose excess energy as heat and fluorescence, or through a number of alternative electron 37 dissipation pathways. 2 Improving photosynthetic efficiency is central to efforts to increase 38 food and/or biofuel yield, and also to realize the biotechnological potential of photosynthetic 39 species. 3 These efforts typically focus on modification of the pigments and proteins involved 40 in light capture, improving the efficiency/specificity of Rubisco or metabolic engineering of 41 product formation downstream of carbon fixation. 4 However, rewiring photosynthesis such 42 that excess reducing potential from light capture is diverted to catalyse the formation of high 43 value products has received little attention. 5 Such a strategy can, in theory, increase the 44 overall efficiency of photosynthetic electron usage by enabling the utilisation of electrons that 45 would otherwise be wasted. 46To increase overall photosynthetic efficiency, we installed the P450 CYP1A1 from 47Rattus norvegicus (brown rat) into Synechococcus PCC 7002 (henceforth Synechococcus) 48 as a new electron-sink in the photosynthetic electron transport chain (Figure 1a). 49Cytochrome P450s are a large and diverse class of monooxygenases that split molecular 50 oxygen (O2), inserting one atom into the substrate and reducing the other to water (Figure 51 1a). CYP1A1 plays a key role in the biotransformation of drugs and other chemical 52 compounds in mammals and has been widely studied. The catalytic activity of CYP1A1 is 53 well-defined and easily assayed, 6 its structure has been resolved at 2.6 Å 7 and it has been 54 expressed in other microbial hosts, allowing comparison of expression and activity levels 55 among species. 8 We designed plasmid pSy21 to express CYP1A1 in Synechococcus. The 56 expression cassette consisted of the cyp1a1 gene, a constitutive phycocyanin promoter from 57 4 Synechocystis PCC 6803, a kanamycin resistance cassette, the rrnB terminator from 58Escherichia coli, and targeting flanks to guide integration to the glpK genomic neutral site 59 (Figure 1b). The cyp1a1 gene was codon-optimized for expression in Synechococcus and 60 the FLAG peptide sequence was added in-frame, along with a 4 x glycine-alanine peptide 61 linker, to the 3' of cyp1a1 to simplify detection, quantification and purification; no other 62 modifications were made to the cyp1a1 sequence. This multipart construct was assemb...

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“…The potential value of combining P450-mediated mono-oxygenation reactions with photosynthesis was first demonstrated in vitro when spinach chloroplasts were brought together with microsomes from yeast expressing a fusion between a P450 from rat (CYP1A1) and a reductase. This mixture supported the light-driven conversion of the P450 substrate 7-ethoxycoumarin into 7-hydroxycoumarin [ 100 ]. More recently, it was shown in vitro that electrons supplied by photosystem I purified from barley could be transferred with high efficiency to a P450 (CYP79A1) from Sorghum bicolor via ferredoxin, thus eliminating the need for an NADPH recycling system and a reductase [ 101 ].…”

Section: Chloroplast Functionsmentioning

confidence: 80%

“…This mixture supported the light-driven conversion of the P450 substrate 7-ethoxycoumarin into 7-hydroxycoumarin [100]. More recently, it was shown in vitro that electrons supplied by photosystem I purified from barley could be transferred with high efficiency to a P450 (CYP79A1) from Sorghum bicolor via ferredoxin, thus eliminating the need for an NADPH recycling system and a reductase [101].…”

Section: Chloroplast Functionsmentioning

confidence: 99%

Chloroplast evolution, structure and functions

Jensen¹,

Leister²

2014

F1000Prime Rep

115

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In this review, we consider a selection of recent advances in chloroplast biology. These include new findings concerning chloroplast evolution, such as the identification of Chlamydiae as a third partner in primary endosymbiosis, a second instance of primary endosymbiosis represented by the chromatophores found in amoebae of the genus Paulinella, and a new explanation for the longevity of captured chloroplasts (kleptoplasts) in sacoglossan sea slugs. The controversy surrounding the three-dimensional structure of grana, its recent resolution by tomographic analyses, and the role of the CURVATURE THYLAKOID1 (CURT1) proteins in supporting grana formation are also discussed. We also present an updated inventory of photosynthetic proteins and the factors involved in the assembly of thylakoid multiprotein complexes, and evaluate findings that reveal that cyclic electron flow involves NADPH dehydrogenase (NDH)- and PGRL1/PGR5-dependent pathways, both of which receive electrons from ferredoxin. Other topics covered in this review include new protein components of nucleoids, an updated inventory of the chloroplast proteome, new enzymes in chlorophyll biosynthesis and new candidate messengers in retrograde signaling. Finally, we discuss the first successful synthetic biology approaches that resulted in chloroplasts in which electrons from the photosynthetic light reactions are fed to enzymes derived from secondary metabolism.

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Photo-induced activation of cytochrome P450/reductase fusion enzyme coupled with spinach chloroplasts

Cited by 14 publications

References 3 publications

Advances in Phytoremediation

Advances in Phytoremediation

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

Chloroplast evolution, structure and functions

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