Insight into Isoprenoid Biosynthesis by Functional Analysis of Isoprenyl Diphosphate Synthases from <i>Mycobacterium vanbaalenii</i> and <i>Mycobacterium tuberculosis</i>

Abe, Tohru; Ozaki, Sadamu; Ueda, Daijiro; Sato, Tsutomu

doi:10.1002/cbic.202000235

Cited by 6 publications

(21 citation statements)

References 27 publications

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“…29 as described below. After culturing as in the case of pColdTF-BmeTC WT , cells expressing the recombinant protein were harvested by centrifugation and disrupted by sonication in buffer B [20 mM Tris-HCl (pH 7.9) and 300 mM NaCl] (30 mL/L cultured cells) containing 10 mM imidazole and 0.1% Tween 80 at 4 °C 29 . The homogenate was centrifuged at 18,270 × g for 20 min to prepare the supernatant containing soluble Histagged fusion protein, which was loaded into a Ni-NTA agarose column (0.2 mL; Qiagen, Hilden, Germany), followed by washing with 10 mL of buffer B containing 10 mM imidazole and then by 12 mL of buffer B containing 50 mM imidazole and 0.1% Tween 80 29 .…”

Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

“…After culturing as in the case of pColdTF-BmeTC WT , cells expressing the recombinant protein were harvested by centrifugation and disrupted by sonication in buffer B [20 mM Tris-HCl (pH 7.9) and 300 mM NaCl] (30 mL/L cultured cells) containing 10 mM imidazole and 0.1% Tween 80 at 4 °C 29 . The homogenate was centrifuged at 18,270 × g for 20 min to prepare the supernatant containing soluble Histagged fusion protein, which was loaded into a Ni-NTA agarose column (0.2 mL; Qiagen, Hilden, Germany), followed by washing with 10 mL of buffer B containing 10 mM imidazole and then by 12 mL of buffer B containing 50 mM imidazole and 0.1% Tween 80 29 . The purified protein was eluted with 3 mL buffer B containing 250 mM imidazole and 0.1% Tween 80, and buffer-exchanged into 3 mL buffer C [50 mM Tris-HCl (pH 7.5), 2.5 mM dithiothreitol, 1 mM EDTA, 300 mM NaCl and 0.1% Tween-80] by gel-filtration chromatography using a Sephadex G-10 column (GE Healthcare, Pittsburgh, PA, USA) 29 .…”

Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

“…The homogenate was centrifuged at 18,270 × g for 20 min to prepare the supernatant containing soluble Histagged fusion protein, which was loaded into a Ni-NTA agarose column (0.2 mL; Qiagen, Hilden, Germany), followed by washing with 10 mL of buffer B containing 10 mM imidazole and then by 12 mL of buffer B containing 50 mM imidazole and 0.1% Tween 80 29 . The purified protein was eluted with 3 mL buffer B containing 250 mM imidazole and 0.1% Tween 80, and buffer-exchanged into 3 mL buffer C [50 mM Tris-HCl (pH 7.5), 2.5 mM dithiothreitol, 1 mM EDTA, 300 mM NaCl and 0.1% Tween-80] by gel-filtration chromatography using a Sephadex G-10 column (GE Healthcare, Pittsburgh, PA, USA) 29 . The expression and purification of BmeTC X were analyzed via 10% SDS-PAGE ( Supplementary Fig.…”

Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

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Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Yamabe¹,

Kawagoe²,

Okuno³

et al. 2020

Sci Rep

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Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, “ambrein synthase,” for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8–15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid β-induced apoptosis, were discovered using the synthesized ambrein.

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Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

Section: Analysis Of Bmetc X Products Using Purified Enzymesmentioning

confidence: 99%

See 1 more Smart Citation

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Yamabe¹,

Kawagoe²,

Okuno³

et al. 2020

Sci Rep

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“…Three E ‐allylic compounds (GPP, FPP and GGPP), the substrates for Z ‐IDSs, are biosynthesized from dimethylallyl diphosphate (DMAPP, C 5 ) by a single enzyme, an E ‐IDS of Mvan_3536 (Fig. 2F) [39]. However, although we hypothesized that Z,E ‐mixed heptaprenyl reductase (HepR), which reduces Z,E ‐mixed prenyl groups, may be involved in the biosynthesis of C 35 PP‐G (Fig.…”

Section: Introductionmentioning

confidence: 99%

Identification and functional analysis of a new type of Z,E‐mixed prenyl reductase from mycobacteria

et al. 2022

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Isoprenoids with reduced Z,E-mixed prenyl groups are found in various organisms. To date, only polyprenol reductases (PR-Dol) involved in dolichol biosynthesis have been identified as enzymes capable of reducing Z,Emixed prenyl groups. Although C 35 -isoprenoids with reduced Z,E-mixed prenyl groups are found in mycobacteria, Z,E-mixed heptaprenyl reductase (HepR) remains unidentified. In the present study, the identification and functional analysis of HepR was performed. No PR-Dol homolog gene was detected in the genome of Mycolicibacterium vanbaalenii. However, a homolog of geranylgeranyl reductase (GGR), which reacts with an all-E prenyl group as a substrate, was encoded in the genome; thus, we analyzed it as a HepR candidate. In vitro enzymatic assay and in vivo gene suppression analysis identified the GGR homolog as HepR and revealed that HepR catalyzes the reduction of xand E-prenyl units in Z,E-mixed heptaprenyl diphosphates, and C 35 -isoprenoids are mainly biosynthesized using E,E,E-geranylgeranyl diphosphate as a precursor. Thus, it was demonstrated that the Z,E-mixed prenyl reductase family exists in the GGR homologs. To the best of our knowledge, this is the first identification of a new type of Z,E-mixed prenyl reductase with no sequence homology to PR-Dol. The substrate specificity of HepR significantly differed from that of GGR, suggesting that it is a new enzyme. HepR homologs are widely distributed in mycobacterial genomes, and lipid analysis suggests that many strains, including pathogenic species, produce HepR metabolites. The discovery of this new enzyme will promote further research on Z,E-mixed isoprenoids.

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“…Thus, the protein rather than the substrate was found to be the contextual virulent factor [ 26 ]. Development of inhibitors for the biosynthesis of menaquinones or other isoprene-derivatives has been explored as potential treatments [ 27 , 28 ].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of Naphthoquinone Derivatives: Menaquinones, Lipoquinones and Other Vitamin K Derivatives

Braasch-Turi

Crans

2020

Molecules

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Menaquinones are a class of isoprenoid molecules that have important roles in human biology and bacterial electron transport, and multiple methods have been developed for their synthesis. These compounds consist of a methylnaphthoquinone (MK) unit and an isoprene side chain, such as found in vitamin K1 (phylloquinone), K2, and other lipoquinones. The most common naturally occurring menaquinones contain multiple isoprene units and are very hydrophobic, rendering it difficult to evaluate the biological activity of these compounds in aqueous assays. One way to overcome this challenge has been the application of truncated MK-derivatives for their moderate solubility in water. The synthesis of such derivatives has been dominated by Friedel-Crafts alkylation with BF3∙OEt2. This attractive method occurs over two steps from commercially available starting materials, but it generally produces low yields and a mixture of isomers. In this review, we summarize reported syntheses of both truncated and naturally occurring MK-derivatives that encompass five different synthetic strategies: Nucleophilic ring methods, metal-mediated reactions, electrophilic ring methods, pericyclic reactions, and homologation and side chain extensions. The advantages and disadvantages of each method are discussed, identifying methods with a focus on high yields, regioselectivity, and stereochemistry leading to a detailed overview of the reported chemistry available for preparation of these compounds.

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Insight into Isoprenoid Biosynthesis by Functional Analysis of Isoprenyl Diphosphate Synthases from Mycobacterium vanbaalenii and Mycobacterium tuberculosis

Cited by 6 publications

References 27 publications

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Identification and functional analysis of a new type of Z,E‐mixed prenyl reductase from mycobacteria

Synthesis of Naphthoquinone Derivatives: Menaquinones, Lipoquinones and Other Vitamin K Derivatives

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