Structural basis of stereospecific reduction by quinuclidinone reductase

Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Kumashiro, Shoko; Nagai, Takahiro; Urano, Nobuyuki; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu

doi:10.1186/2191-0855-4-6

Cited by 7 publications

(9 citation statements)

References 36 publications

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“…whose structures have been solved, and which contain a large, extended N-terminal region, include a quinuclidinone reductase (RrQR) from Rhodotorula rubra (24) and an S-specific carbonyl reductase from Candida parapsilosis (25). There was no electron density for the first six amino acid residues of RrQR, indicating a flexible region.…”

Section: Characterization Of 20␤-hydroxysteroid Dehydrogenasementioning

confidence: 99%

Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

Doden

Pollet

Mythen

et al. 2019

Journal of Biological Chemistry

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Edited by Chris Whitfield Anaerobic bacteria inhabiting the human gastrointestinal tract have evolved various enzymes that modify host-derived steroids. The bacterial steroid-17,20-desmolase pathway cleaves the cortisol side chain, forming pro-androgens predicted to impact host physiology. Bacterial 20␤-hydroxysteroid dehydrogenase (20␤-HSDH) regulates cortisol side-chain cleavage by reducing the C-20 carboxyl group on cortisol, yielding 20␤-dihydrocortisol. Recently, the gene encoding 20␤-HSDH in Butyricicoccus desmolans ATCC 43058 was reported, and a nonredundant protein search yielded a candidate 20␤-HSDH gene in Bifidobacterium adolescentis strain L2-32. B. adolescentis 20␤-HSDH could regulate cortisol side-chain cleavage by limiting pro-androgen formation in bacteria such as Clostridium scindens and 21-dehydroxylation by Eggerthella lenta. Here, the putative B. adolescentis 20␤-HSDH was cloned, overexpressed, and purified. 20␤-HSDH activity was confirmed through whole-cell and pure enzymatic assays, and it is specific for cortisol. Next, we solved the structures of recombinant 20␤-HSDH in both the apo-and holo-forms at 2.0 -2.2 Å resolutions, revealing close overlap except for rearrangements near the active site. Interestingly, the structures contain a large, flexible N-terminal region that was investigated by gel-filtration chromatography and CD spectroscopy. This extended N terminus is important for protein stability because deletions of varying lengths caused structural changes and reduced enzymatic activity. A nonconserved extended N terminus was also observed in several short-chain dehydrogenase/reductase family members. B. adolescentis strains capable of 20␤-HSDH activity could alter glucocorticoid metabolism in the gut and thereby serve as potential probiotics for the management of androgen-dependent diseases.

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Section: Characterization Of 20␤-hydroxysteroid Dehydrogenasementioning

confidence: 99%

Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

Doden

Pollet

Mythen

et al. 2019

Journal of Biological Chemistry

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“…20) However, the enzyme shows a 100 times higher substrate-binding affinity as compared with RrQR, indicating that the substrate-binding site of AtQR is modified to permit additional interaction(s). AtQR adopts the small substrate-binding site, which is formed by the arrangements of additional residues at the space exposed to solvent in the structure of RrQR.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…2(C) and (D)). 19,20) In the structure of RrQR, Ser166 forms a hydrogen bond with the carbonyl oxygen of 3-quinuclidinone. Hydride is transferred to the carbonyl carbon of the substrate from the nicotinamide ring of NADPH.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…X-ray crystallographic studies of these enzymes have revealed the unique mechanisms underlying their strict stereoselectivity and enhanced substrate-binding affinity upon cofactor binding. 19,20) Both quinuclidinone reductases share a core (or large) domain composed of a typical Rossmann fold with a seven-stranded parallel β-sheet and seven α-helices, and form a tightly associated homotetramer. 19,20) Although the cofactor-binding site is located at the core domain in both reductases, RrQR and AtQR exhibit different cofactor selectivities toward NADPH and NADH, respectively.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

“…19,20) Both quinuclidinone reductases share a core (or large) domain composed of a typical Rossmann fold with a seven-stranded parallel β-sheet and seven α-helices, and form a tightly associated homotetramer. 19,20) Although the cofactor-binding site is located at the core domain in both reductases, RrQR and AtQR exhibit different cofactor selectivities toward NADPH and NADH, respectively. The selectivity is defined by the residues surrounding the ribose ring of the adenosine moiety ( Fig.…”

Section: Structural Basis For the Stereoselectivity Of Quinuclidinmentioning

confidence: 99%

See 2 more Smart Citations

Structural analysis of enzymes used for bioindustry and bioremediation

Tanokura

Miyakawa

Guan

et al. 2015

Bioscience, Biotechnology, and Biochemistry

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Microbial enzymes have been widely applied in the large-scale, bioindustrial manufacture of food products and pharmaceuticals due to their high substrate specificity and stereoselectivity, and their effectiveness under mild conditions with low environmental burden. At the same time, bioremedial techniques using microbial enzymes have been developed to solve the problem of industrial waste, particularly with respect to persistent chemicals and toxic substances. And finally, structural studies of these enzymes have revealed the mechanistic basis of enzymatic reactions, including the stereoselectivity and binding specificity of substrates and cofactors. The obtained structural insights are useful not only to deepen our understanding of enzymes with potential bioindustrial and/or bioremedial application, but also for the functional improvement of enzymes through rational protein engineering. This review shows the structural bases for various types of enzymatic reactions, including the substrate specificity accompanying cofactor-controlled and kinetic mechanisms.

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Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use

Kataoka

Miyakawa

Shimizu

et al. 2016

Appl Microbiol Biotechnol

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Biocatalysts (enzymes) have many advantages as catalysts for the production of useful compounds as compared to chemical catalysts. The stereoselectivity of the enzymes is one advantage, and thus the stereoselective production of chiral compounds using enzymes is a promising approach. Importantly, industrial application of the enzymes for chiral compound production requires the discovery of a novel useful enzyme or enzyme function; furthermore, improving the enzyme properties through protein engineering and directed evolution approaches is significant. In this review, the significance of several enzymes showing stereoselectivity (quinuclidinone reductase, aminoalcohol dehydrogenase, old yellow enzyme, and threonine aldolase) in chiral compound production is described, and the improvement of these enzymes using protein engineering and directed evolution approaches for further usability is discussed. Currently, enzymes are widely used as catalysts for the production of chiral compounds; however, for further use of enzymes in chiral compound production, improvement of enzymes should be more essential, as well as discovery of novel enzymes and enzyme functions.

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Structural basis of stereospecific reduction by quinuclidinone reductase

Cited by 7 publications

References 36 publications

Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32

Structural analysis of enzymes used for bioindustry and bioremediation

Enzymes useful for chiral compound synthesis: structural biology, directed evolution, and protein engineering for industrial use

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