Properties and evolution of an alcohol dehydrogenase from the Crenarchaeota Pyrobaculum aerophilum

Vitale, Alejandro José; Rosso, Francesco; Barbarisi, Alfonso; Labella, Tullio; D’Auria, Sabato

doi:10.1016/j.gene.2010.04.004

Cited by 9 publications

(14 citation statements)

References 32 publications

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“…All enzymes belonging to the MDR superfamily use NAD(H) or NADP(H) as a cofactor (22), and catalyze oxidation or reduction reactions biologically (23). These characteristics support the requirement of NADPH as a cofactor for the reduction of curcumin/dihydrocurcumin by CurA.…”

Section: Discussionmentioning

confidence: 96%

Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism

Hassaninasab

Hashimoto

Tomita‐Yokotani

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

230

155

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Polyphenol curcumin, a yellow pigment, derived from the rhizomes of a plant (Curcuma longa Linn) is a natural antioxidant exhibiting a variety of pharmacological activities and therapeutic properties. It has long been used as a traditional medicine and as a preservative and coloring agent in foods. Here, curcumin-converting microorganisms were isolated from human feces, the one exhibiting the highest activity being identified as Escherichia coli. We are thus unique in discovering that E. coli was able to act on curcumin. The curcumin-converting enzyme was purified from E. coli and characterized. The native enzyme had a molecular mass of about 82 kDa and consisted of two identical subunits. The enzyme has a narrow substrate spectrum, preferentially acting on curcumin. The microbial metabolism of curcumin by the purified enzyme was found to comprise a two-step reduction, curcumin being converted NADPHdependently into an intermediate product, dihydrocurcumin, and then the end product, tetrahydrocurcumin. We named this enzyme "NADPH-dependent curcumin/dihydrocurcumin reductase" (CurA). The gene (curA) encoding this enzyme was also identified. A homology search with the BLAST program revealed that a unique enzyme involved in curcumin metabolism belongs to the mediumchain dehydrogenase/reductase superfamily. microbial screening | bioconversion S ince the dawn of civilization, natural compounds have been used as a source of medicine and foods based on their tremendous biological activities. One of the natural compounds that has been documented as having been used as a spice and a pigment since 1900 B.C. is curcumin (1). Curcumin, defined as a bis-α, β-unsaturated β-diketone (Fig. S1), has been found exclusively in the roots of Curcuma longa Linn (Zingiberaceae) (2). Curcumin has many different applications because it has a surprisingly wide variety of beneficial uses, including food coloration, cosmetics utility, and fabric dying. In addition, curcumin has a wide range of medical properties, including antitumor, antiinflammatory, antioxidant, anticancer, and analgesic uses (3). Recently, curcumin has been used in the treatment of Alzheimer's disease (4) and malaria (5), and improvement of wound healing (6). Curcumin in the diet is partially absorbed in the intestine (7). A considerable portion of the ingested curcumin reaches the cecum and colon, where a large population of indigenous bacteria exists. Although analysis of curcumin metabolism and biosynthesis would lead to the discovery of other unknown advantages of this compound, there has neither been purification of enzymes involved in the metabolic pathway for curcumin nor identification of their genes in living organisms. In addition, no information is available regarding the effect of the intestinal bacteria on curcumin metabolism.Studies on such curcumin-converting microorganisms and functional analyses of the curcumin-converting enzymes and genes would facilitate clarification of the metabolism of curcumin. In the present article, we report the isolation of cur...

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Section: Discussionmentioning

confidence: 96%

Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism

Hassaninasab

Hashimoto

Tomita‐Yokotani

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

230

155

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“…The wild-type PyAeADHII was cloned and purified as described elsewhere [9]. PyAeADHII activity was assayed at 37°C by measuring the change in absorbance of NADPH at 340 nm, using a Cary 1E spectrophotometer equipped with a Peltier effect-controlled temperature cuvette holder.…”

Section: Methodsmentioning

confidence: 99%

“…An HTS assay was developed that utilized the fluorescence of NADPH (λ Ex 340 nm/λ Em 450 nm), a cofactor consumed in the reduction of α-tetralone by PyAeADHII [9], as a read-out of enzyme activity. Compounds that inhibit PyAeADHII would be expected to decrease NADPH consumption by the enzyme, and thus the fluorescence at 450 nm would be greater than uninhibited enzyme.…”

Section: Methodsmentioning

confidence: 99%

“…Previous experiments identified α-tetralone as a substrate of PyAeADHII [9]. These experiments were conducted at 70°C using a spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

“…This ADH is characterized as belonging to the medium-chain dehydrogenase/reductase (MDR) superfamily, with a size of 330 residues and a structural Zn 2+ binding site made up of four closely spaced cysteine residues localized in a lobe at the periphery of the catalytic domain [8]. However, previous studies have revealed that PyAeADHII has peculiar characteristics because the enzyme lacked activity on most standard compounds used to test the activity of ADHs, and was active only when α-tetralone was used as a substrate [9]. Moreover, sequence alignment of PyAeADHII with sequences of well characterized ADHs, such as horse liver ADH (HLADH 6ADH_B) [10], Saccharomyces cerevisiae ADH (YADH CAA91579) [11], [12] and Sulfolobus solfataricus ADH (SsADH CAA87591) [13] showed that the PyAeADHII lacks key residues involved in the catalytic Zn 2+ binding (e.g.…”

Section: Introductionmentioning

confidence: 99%

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Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

et al. 2013

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In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn2+) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn2+ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn2+ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.

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Application of Two‐dimensional Correlation Spectroscopy in Protein Research

Jung

Czarnik-Matusewicz

et al. 2015

Encyclopedia of Analytical Chemistry

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Properties and evolution of an alcohol dehydrogenase from the Crenarchaeota Pyrobaculum aerophilum

Cited by 9 publications

References 32 publications

Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism

Discovery of the curcumin metabolic pathway involving a unique enzyme in an intestinal microorganism

Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

Application of Two‐dimensional Correlation Spectroscopy in Protein Research

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