Bacterial Phytoene Synthase:  Molecular Cloning, Expression, and Characterization of<i>Erwinia herbicola</i>Phytoene Synthase

Iwata‐Reuyl, Dirk; Math, Shivanand K.; Desai, Shrivallabh B.; Poulter, C. Dale

doi:10.1021/bi0206614

Cited by 27 publications

(39 citation statements)

References 48 publications

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“…VKIRK is the most flexible part of human SQS in its crystal structure and is thought to be stabilized by NADPH binding (32). A related enzyme, phytoene synthase (PS), converts geranylgeranyl diphosphate to phytoene (13,28). SQS and PS both catalyze the cyclopropanation and rearrangement reactions shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

Lee

Poulter

2008

J Bacteriol

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Squalene synthase (SQS) is a bifunctional enzyme that catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to give presqualene diphosphate (PSPP) and the subsequent rearrangement of PSPP to squalene. These reactions constitute the first pathway-specific steps in hopane biosynthesis in Bacteria and sterol biosynthesis in Eukarya. The genes encoding SQS were isolated from the hopane-producing bacteria Thermosynechococcus elongatus BP-1, Bradyrhizobium japonicum, and Zymomonas mobilis and cloned into an Escherichia coli expression system. The expressed proteins with a His 6 tag were found exclusively in inclusion bodies when no additives were used in the buffer. After extensive optimization, soluble recombinant T. elongatus BP-1 SQS was obtained when cells were disrupted and purified in buffers containing glycerol. The recombinant B. japonicum and Z. mobilis SQSs could not be solubilized under any of the expression and purification conditions used. Purified T. elongatus His 6 -SQS gave a single band at 42 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and molecular ion at m/z 41886 by electrospray mass spectrometry. Incubation with FPP and NADPH gave squalene as the sole product. Incubation of the enzyme with [14 C]FPP in the absence of NADPH gave PSPP. The enzyme requires Mg 2؉ for activity, has an optimum pH of 7.6, and is strongly stimulated by detergent. Under optimal conditions, the K m of FPP is 0.97 ؎ 0.10 M and the k cat is 1.74 ؎ 0.04 s ؊1 . Zaragozic acid A, a potent inhibitor of mammalian, fungal, and Saccharomyces cerevisiae SQSs, also inhibited recombinant T. elongatus BP-1 SQS, with a 50% inhibitory concentration of 95.5 ؎ 13.6 nM.Squalene synthase ([SQS] farnesyl diphosphate:farnesyl diphosphate transferase; EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form a c1Ј-2-3-linked triterpene intermediate, presqualene diphosphate (PSPP), and the subsequent NADPH-dependent rearrangement and reduction of PSPP to produce squalene (SQ), as outlined in Fig. 1 (36, 37). These transformations are the first pathway-specific reactions in the hopanoid biosynthetic pathway in Bacteria (7,33,42,48) and the sterol pathway in Eukarya (37). Studies of yeast SQS show that substrate addition is ordered with two molecules of FPP added first, followed by NADPH (24, 39). When NADPH is present in the buffer, PSPP is converted directly to SQ without dissociating from the active site. Formation of PSPP or a prior conformational change in SQS is the rate-limiting step in the overall conversion of FPP to SQ. PSPP is formed and released when NADPH is absent. Under these conditions, SQS catalyzes a slow "solvolysis" of PSPP to give triterpene alcohols and hydrocarbons with irregular isoprenoid skeletons (14).SQS has been cloned from a variety of eukaryotes, including fungal (15, 21, 57), protozoan (45), rat (22), mouse (12), human (41, 50), and plant sources (27). The eukaryotic enzyme is associated with membranes, where it appears to be anchored by a h...

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Section: Resultsmentioning

confidence: 99%

“…Geranyl diphosphate (GPP) and FPP were synthesized by the procedure of Davisson et al (8). [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]isopentenyl diphosphate (IPP) and [1][2][3] H]FPP were purchased from GE Healthcare. General methods.…”

Section: Methodsmentioning

confidence: 99%

Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

Lee

Poulter

2008

J Bacteriol

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“…NRC-1 (19), Haloarcula marismortui (18), Halobacterium salinarum (22), uncultured marine bacterium 66A03 (16), and uncultured marine bacterium HF10 49E08 (21). ␤-Carotene biosynthetic genes crtE, crtB, crtI, crtY, ispA, and idi encode the enzymes necessary for the synthesis of ␤-carotene from isopentenyl diphosphate, and the Idi, IspA, CrtE, CrtB, CrtI, and CrtY proteins have been characterized in vitro (23)(24)(25)(26)(27)(28). Blh protein has been proposed to catalyze or regulate the conversion of ␤-carotene to retinal (29, 30), but there is no direct proof of the enzymatic activity.…”

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confidence: 99%

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Kim

Yeom

et al. 2009

Journal of Biological Chemistry

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Codon optimization was used to synthesize the blh gene from the uncultured marine bacterium 66A03 for expression in Escherichia coli. The expressed enzyme cleaved ␤-carotene at its central double bond (15,15) to yield two molecules of all-transretinal. The molecular mass of the native purified enzyme was ϳ64 kDa as a dimer of 32-kDa subunits. The K m , k cat , and k cat /K m values for ␤-carotene as substrate were 37 M, 3.6 min ؊1 , and 97 mM ؊1 min ؊1 , respectively. The enzyme exhibited the highest activity for ␤-carotene, followed by ␤-cryptoxanthin, ␤-apo-4-carotenal, ␣-carotene, and ␥-carotene in decreasing order, but not for ␤-apo-8-carotenal, ␤-apo-12-carotenal, lutein, zeaxanthin, or lycopene, suggesting that the presence of one unsubstituted ␤-ionone ring in a substrate with a molecular weight greater than C 35 seems to be essential for enzyme activity. The oxygen atom of retinal originated not from water but from molecular oxygen, suggesting that the enzyme was a ␤-carotene 15,15-dioxygenase. Although the Blh protein and ␤-carotene 15,15-monooxygenases catalyzed the same biochemical reaction, the Blh protein was unrelated to the mammalian ␤-carotene 15,15-monooxygenases as assessed by their different properties, including DNA and amino acid sequences, molecular weight, form of association, reaction mechanism, kinetic properties, and substrate specificity. This is the first report of in vitro characterization of a bacterial ␤-carotene-cleaving enzyme.Vitamin A (retinol) is a fat-soluble vitamin and important for human health. In vivo, the cleavage of ␤-carotene to retinal is an important step of vitamin A synthesis. The cleavage can proceed via two different biochemical pathways (1, 2). The major pathway is a central cleavage catalyzed by mammalian ␤-carotene 15,15Ј-monooxygenases (EC 1.14.99.36). ␤-Carotene is cleaved by the enzyme symmetrically into two molecules of alltrans-retinal, and retinal is then converted to vitamin A in vivo (3-5). The second pathway is an eccentric cleavage that occurs at double bonds other than the central 15,15Ј-double bond of ␤-carotene to produce ␤-apo-carotenals with different chain lengths, which are catalyzed by carotenoid oxygenases from mammals, plants, and cyanobacteria (6). These ␤-apo-carotenals are degraded to one molecule of retinal, which is subsequently converted to vitamin A in vivo (2).␤-Carotene 15,15Ј-monooxygenase was first isolated as a cytosolic enzyme by identifying the product of ␤-carotene cleavage as retinal (7). The characterization of the enzyme and the reaction pathway from ␤-carotene to retinal were also investigated (4, 8). The enzyme activity has been found in mammalian intestinal mucosa, jejunum enterocytes, liver, lung, kidney, and brain (5, 9, 10). Molecular cloning, expression, and characterization of ␤-carotene 15,15Ј-monooxygenase have been reported from various species, including chickens (11), fruit flies (12), humans (13), mice (14), and zebra fishes (15).Other proteins thought to convert ␤-carotene to retinal include bacterioopsin-r...

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“…1). (Most phytoene synthases produce the 15Z isomer of phytoene [87].) Different types and levels of modification of this C 40 backbone account for the majority of known carotenoids.…”

Section: Overview Of Carotenoid Biosynthetic Pathwaysmentioning

confidence: 99%

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Umeno

Tobias

Arnold

2005

Microbiol Mol Biol Rev

195

148

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Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed

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Bacterial Phytoene Synthase: Molecular Cloning, Expression, and Characterization ofErwinia herbicolaPhytoene Synthase

Cited by 27 publications

References 48 publications

Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

Cloning, Solubilization, and Characterization of Squalene Synthase from Thermosynechococcus elongatus BP-1

In Vitro Characterization of a Recombinant Blh Protein from an Uncultured Marine Bacterium as a β-Carotene 15,15′-Dioxygenase

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

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