Molecular Cloning and Characterization of Hemolymph 3-Dehydroecdysone 3β-Reductase from the Cotton Leafworm,Spodoptera littoralis

Chen, Jianhua; Turner, Philip C.; Rees, Huw H.

doi:10.1074/jbc.274.15.10551

Cited by 36 publications

(25 citation statements)

References 28 publications

(18 reference statements)

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“…However, a few members of the family can use both cofactors. For example, xylose reductases from the yeasts P. tannophilus (6) and P. stipitis (49), an ADR from the yeast C. tenuis (36), a 3␣-hydroxysteroid dehydrogenase from rat liver (40), and a 3-dehydroecdysone 3␤-reductase cloned from Spodoptera littoralis (9,10) are thought to have dual cofactor specificity. As far as we are aware, no members of the ADR family are specific for NADH only or show higher affinity for NADH than for NADPH.…”

Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Lee¹,

Kim²,

Ryu

et al. 2003

Appl Environ Microbiol

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Erythritol biosynthesis is catalyzed by erythrose reductase, which converts erythrose to erythritol. Erythrose reductase, however, has never been characterized in terms of amino acid sequence and kinetics. In this study, NAD(P)H-dependent erythrose reductase was purified to homogeneity from Candida magnoliae KFCC 11023 by ion exchange, gel filtration, affinity chromatography, and preparative electrophoresis. The molecular weights of erythrose reductase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography were 38,800 and 79,000, respectively, suggesting that the enzyme is homodimeric. Partial amino acid sequence analysis indicates that the enzyme is closely related to other yeast aldose reductases. C. magnoliae erythrose reductase catalyzes the reduction of various aldehydes. Among aldoses, erythrose was the preferred substrate (K m ‫؍‬ 7.9 mM; k cat /K m ‫؍‬ 0.73 mM ؊1 s ؊1 ). This enzyme had a dual coenzyme specificity with greater catalytic efficiency with NADH (k cat /K m ‫؍‬ 450 mM ؊1 s ؊1 ) than with NADPH (k cat /K m ‫؍‬ 5.5 mM ؊1 s ؊1 ), unlike previously characterized aldose reductases, and is specific for transferring the 4-pro-R hydrogen of NADH, which is typical of members of the aldo/keto reductase superfamily. Initial velocity and product inhibition studies are consistent with the hypothesis that the reduction proceeds via a sequential ordered mechanism. The enzyme required sulfhydryl compounds for optimal activity and was strongly inhibited by Cu 2؉ and quercetin, a strong aldose reductase inhibitor, but was not inhibited by aldehyde reductase inhibitors and did not catalyze the reduction of the substrates for carbonyl reductase. These data indicate that the C. magnoliae erythrose reductase is an NAD(P)H-dependent homodimeric aldose reductase with an unusual dual coenzyme specificity.Erythritol, a four-carbon polyol, is widely distributed in nature (16). Like most other polyols, erythritol is a metabolite or storage compound and is found in seaweed, mushrooms, and fruits. It is a noncaloric, noncariogenic sweetener that is safe for diabetics (35). Erythritol has about 70% of the sweetness of sucrose in a 10% (wt/vol) solution and a very high negative heat capacity, providing a strong cooling effect when dissolved (16). Erythritol can be synthesized from dialdehyde starch by a high-temperature chemical reaction in the presence of a nickel catalyst (39). This process has not been industrialized because of its low efficiency. Erythritol also can be produced by microbial methods that utilize osmophilic yeasts and some bacteria (42, 48), and it has been produced commercially by using a mutant of Aureobasidium that produces erythritol in high yield (44% [wt/wt] glucose) (20). Recently, a high-erythritol-producing yeast strain was isolated from honeycombs and the new isolate was identified as Candida magnoliae KFCC 11023 (44). This strain can produce erythritol in high yield (43% [wt/wt] glucose) when cultured appropriately (42, 44).There are many repor...

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Section: Discussionmentioning

confidence: 99%

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Lee¹,

Kim²,

Ryu

et al. 2003

Appl Environ Microbiol

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“…According to previous reports, 3DE-3b-reductase can convert the 3-dehydro-ecdysteroid to ecdysteroid (22)(23)(24)(25)(26). If the expression level of the 3DE-3b-reductase gene was downregulated, its enzymatic products might also decrease.…”

Section: De-3b-reductase Gene Is Required For the Silkworm To Defendmentioning

confidence: 99%

“…However, cotton leafworm (Spodoptera littoralis) 3DE-3b-reductase protein has a signal peptide (26). Therefore, we used RACE to obtain the full length of the silkworm 3DE-3b-reductase gene.…”

Section: Effects Of 20e On Silkworm Immunitymentioning

confidence: 99%

“…In immature stages of most lepidopteran species, 3DE is the major product of the prothoracic glands. After release from the glands, 3DE is rapidly reduced to ecdysone by 3DE-3b-reductase in hemolymph, and then the ecdysone is converted into 20E by 20-hydroxylase in the target tissues (22)(23)(24)(25)(26). 3DE-3b-reductase is considered to be an important enzyme in the biosynthesis of the ecdysone (27).…”

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confidence: 99%

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Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Sun

Shen

Zhou

et al. 2016

The Journal of Immunology

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Although recent studies have demonstrated that 20-hydroxyecdysone (20E), one of the two most important hormones for development, could promote the insect innate immune response, how insects regulate 20E titer to affect the immunity after suffering pathogen attack remains unknown. In this study, to our knowledge, we first found that 20E titer was significantly elevated after bacterial infection in the domesticated silkworm, Bombyx mori. Furthermore, the elevated 20E enhanced the silkworm innate immune system against invading bacteria via ecdysone receptor. During immune response, the expression of the silkworm 3-dehydroecdysone-3β-reductase (3DE-3β-reductase) that converts 3DE released from prothoracic glands into ecdysone was induced. RNA interference experiments suggested that 3DE-3β-reductase is essential to upregulate the 20E titer after bacterial infection. The rescue experiments showed that injection with the recombinant 3DE-3β-reductase protein can significantly elevate the 20E concentration and modulate the expressions of the silkworm immune-related genes. Taken together, 20E titer determined by 3DE-3β-reductase enhances the silkworm defense against the bacterial infection. Thus, our findings reveal an important role of the 20E synthesis pathway from 3DE in enhancing the silkworm immune response and have profound implications for the understanding of interaction mechanisms between insect hormone and immunity.

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“…As part of our studies aimed at elucidating the regulation of ecdysteroid titer, including the reactions involved in ecdysteroid inactivation in S. littoralis, we have cloned and characterized the cDNA encoding the 3DE 3␤-reductase (hemolymph) (20) and 3DE 3␣-reductase (Malpighian tubules) (16). Furthermore, we have purified ecdysone oxidase from S. littoralis midgut plus attached Malpighian tubules, and evidence sug-gests that the native enzyme consists of a trimer with apparent molecular mass of ϳ190 kDa and subunit molecular mass of ϳ64 kDa (21).…”

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confidence: 99%

Regulation of Ecdysteroid Signaling: Cloning and Characterization of Ecdysone Oxidase

Takeuchi

Chen

O’Reilly³

et al. 2001

Journal of Biological Chemistry

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One route of inactivation of ecdysteroids in insects involves ecdysone oxidase-catalyzed conversion into 3-dehydroecdysteroid followed by irreversible reduction by 3-dehydroecdysone 3␣-reductase to 3-epiecdysone. We have purified from Spodoptera littoralis the first ecdysone oxidase and subjected it to limited amino acid sequencing. A reverse-transcriptase polymerase chain reaction-based approach has been used to clone the cDNA (2.8 kilobases) encoding this 65-kDa protein.Northern blotting showed that the mRNA transcript was expressed in midgut during the prepupal stage of the last larval instar at a time corresponding to an ecdysteroid titer peak. Conceptual translation of the ecdysone oxidase cDNA and data base searching revealed that the enzyme is an FAD flavoprotein that belongs to the glucose-methanol-choline oxidoreductase superfamily. Ecdysone oxidase represents the only oxidase in eukaryotic animals known to catalyze oxygen-dependent oxidation of steroids; by contrast, oxidation of steroids in vertebrates occurs via NAD(P) ؉ -linked dehydrogenases. The injection of RH-5992, an ecdysteroid agonist, induced the transcription of ecdysone oxidase, suggesting that ecdysone oxidase is an ecdysteroid-responsive gene. The gene encoding this enzyme, consisting of five exons, has also been isolated. Sequences similar to the binding motifs for Broad-Complex and FTZ-F1 have been found in the 5-flanking region. Southern blotting indicated that ecdysone oxidase is encoded by a singlecopy gene. We have determined the kinetic characteristics of this novel recombinant ecdysone oxidase produced using a baculovirus expression system.

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Molecular Cloning and Characterization of Hemolymph 3-Dehydroecdysone 3β-Reductase from the Cotton Leafworm,Spodoptera littoralis

Cited by 36 publications

References 28 publications

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Purification and Characterization of a Novel Erythrose Reductase from Candida magnoliae

Ecdysone Titer Determined by 3DE-3β-Reductase Enhances the Immune Response in the Silkworm

Regulation of Ecdysteroid Signaling: Cloning and Characterization of Ecdysone Oxidase

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