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

Abstract

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 NADPH-dependently 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 medium-chain dehydrogenase/reductase superfamily.

Fig. 1.

SDS/PAGE of the purified curcumin-converting enzyme. Protein bands were detected by staining with Coomassie brilliant blue. Lane M, marker proteins; lane 1, the purified enzyme (1 μg).

Fig. 2.

The reaction products, dihydrocurcumin (DHC), and tetrahydrocurcumin (THC). (A) LC-ESI-MS spectrum of THC in the positive ion mode. (B) UV-vis spectrum of THC. (C) LC-ESI-MS spectrum of DHC in the positive ion mode. (D) UV-vis spectrum of DHC.

Fig. 3.

Time course of curcumin conversion and generation of products. Curcumin (▲), DHC (●), and THC (■).

Fig. 4.

Curcumin metabolic pathway.

Fig. 5.

CD spectra of the curcumin-converting enzyme and the ellipticity in different pH values. The CD spectra of CurA treated in 10-mM Britton-Robinson buffer pH 6.0 (A) and pH 2.1 (B); 0.1 mg/mL of the purified enzyme was treated at different pH values in 10-mM Britton-Robinson buffer and CD spectra were measured. The mollar ellipticity (θ) was then calculated for the different pHs. θ₂₂₂ was denoted by dotted line. (C) θ₂₂₂ at different pH values. The CD spectra of CurA after the pH was shifted from 6.0 to 6.0 (D) and from 2.1 to 6.0 (E); 0.1 mg/mL of the purified enzyme was treated at different pH values in 10-mM Britton-Robinson buffer and then shifted to the usual pH 6.0 using 40-mM Britton-Robinson buffer (pH 6.0), and then the CD spectrum was measured and θ₂₂₂ was denoted by a dotted line. (F), θ₂₂₂ after the pH had been shifted from various values to 6.0.