Complete enzyme set for chlorophyll biosynthesis in
            <i>Escherichia coli</i>

Chen, Guangyu E.; Canniffe, Daniel P.; Barnett, Samuel F. H.; Hollingshead, Sarah; Brindley, Amanda A.; Vasilev, Cvetelin; Bryant, Donald A.; Hunter, C. Neil

doi:10.1126/sciadv.aaq1407

Cited by 46 publications

(52 citation statements)

References 40 publications

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“…It is possible that a large multi-enzyme assembly could channel photolabile biosynthetic intermediates between active sites, minimizing their exposure to light and oxygen. The heterologous assembly of the Chl biosynthesis pathway in E. coli provides a platform for investigating the physical and mechanistic coupling between pathway enzymes (238). This achievement also demonstrates that, after decades of research, all reactions necessary for the biosynthesis of Chl a have now been identified.…”

Section: The Transformation Of Protoix Into Chls -Chl Amentioning

confidence: 79%

Biosynthesis of the modified tetrapyrroles—the pigments of life

Bryant

Hunter

Warren

2020

Journal of Biological Chemistry

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196

164

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Modified tetrapyrroles are large macrocyclic compounds, consisting of diverse conjugation and metal chelation systems and imparting an array of colors to the biological structures that contain them. Tetrapyrroles represent some of the most complex small molecules synthesized by cells and are involved in many essential processes that are fundamental to life on Earth, including photosynthesis, respiration, and catalysis. These molecules are all derived from a common template through a series of enzyme-mediated transformations that alter the oxidation state of the macrocycle and also modify its size, its side-chain composition, and the nature of the centrally chelated metal ion. The different modified tetrapyrroles include chlorophylls, hemes, siroheme, corrins (including vitamin B12), coenzyme F430, heme d1, and bilins. After nearly a century of study, almost all of the more than 90 different enzymes that synthesize this family of compounds are now known, and expression of reconstructed operons in heterologous hosts has confirmed that most pathways are complete. Aside from the highly diverse nature of the chemical reactions catalyzed, an interesting aspect of comparative biochemistry is to see how different enzymes and even entire pathways have evolved to perform alternative chemical reactions to produce the same end products in the presence and absence of oxygen. Although there is still much to learn, our current understanding of tetrapyrrole biogenesis represents a remarkable biochemical milestone that is summarized in this review.

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Section: The Transformation Of Protoix Into Chls -Chl Amentioning

confidence: 79%

Biosynthesis of the modified tetrapyrroles—the pigments of life

Bryant

Hunter

Warren

2020

Journal of Biological Chemistry

Self Cite

196

164

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“…Geranylgeranyl is incorporated into chlorophyll by ChlG in the second last step. In a landmark paper, expression of ChlDHI and GUN4, ChlM, ChlA1, LPOR, BciB, ChlG and ChlP in E. coli was sufficient for chlorophyll biosynthesis [196], demonstrating that no other enzymes are required in this pathway.…”

Section: Chlorophyll Biosynthesismentioning

confidence: 99%

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

2020

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Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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“…In a recent study, heterologous expression of R. gelatinosus acsF in Escherichia coli demonstrated that cyclase activity could occur in vivo in this non-photosynthetic organism [ 3 ]. While this study suggested that any remaining parts of the enzyme are commonly present in E. coli cells, it did not fully define the essential components necessary for a completely recombinant system where all required partners are present.…”

Section: Resultsmentioning

confidence: 99%

“…Most steps for chlorophyll biosynthesis have been studied using recombinant proteins in defined reactions, but the MPE cyclase is an exception. Instead, present knowledge concerning the cyclase is based on studies using fractionated cell extracts and genetic analyses of bacteria, algae, and plants [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. Two distinct enzymes have been identified that catalyze the cyclase reaction, originally distinguished by the source of the incorporated oxygen.…”

Section: Introductionmentioning

confidence: 99%

“…Fractionation of cell extracts of cucumber ( Cucumis sativus L.) [ 20 , 21 ], C. reinhardtii , Synechocystis [ 22 ], and barley [ 7 , 23 ] revealed that cyclase activity requires both additional soluble and membrane-bound fractions, but all involved components have not been identified. The discovery of Ycf54 and LCAA in Synechocystis [ 24 ] and tobacco Nicotiana tabacum L. [ 2 ], respectively, indicated that this protein affected the cyclase enzyme function in organisms that perform oxygenic photosynthesis, although the protein is not required for cyclase activity by the acsF -encoded aerobic cyclase found in organisms that perform anoxygenic photosynthesis [ 3 ]. Despite the lack of an active recombinant enzyme in vitro, a reaction mechanism of the aerobic cyclase has been proposed.…”

Section: Introductionmentioning

confidence: 99%

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Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

Stuart

Sandström

Youssef

et al. 2020

Plants

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Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.

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Complete enzyme set for chlorophyll biosynthesis in Escherichia coli

Abstract: Escherichia coli has been engineered to produce chlorophyll.

Cited by 46 publications

References 40 publications

Biosynthesis of the modified tetrapyrroles—the pigments of life

Biosynthesis of the modified tetrapyrroles—the pigments of life

Current knowledge and recent advances in understanding metabolism of the model cyanobacteriumSynechocystissp. PCC 6803

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

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