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...

Several general mechanisms of metallocenter biosynthesis have been reported and reviewed, and in all cases, the components or subunits of an apoprotein remain in the final holoprotein. Here, we first discovered that one subunit of an apoenzyme did not remain in the functional holoenzyme. The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 consists of ␤-and ␣-subunits encoded by the nhlBA genes, respectively. An ORF, nhlE, just downstream of nhlBA, was found to be necessary for L-NHase activation. In contrast to the cobaltcontaining L-NHase (holo-L-NHase containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands) derived from nhlBAE, the gene products derived from nhlBA were cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues). We discovered an L-NHase maturation mediator, NhlAE, consisting of NhlE and the cobalt-and oxidized cysteine-containing ␣-subunit of L-NHase. The incorporation of cobalt into L-NHase was shown to depend on the exchange of the nonmodified cobalt-free ␣-subunit of apo-L-NHase with the cobaltcontaining cysteine-modified ␣-subunit of NhlAE. This is a posttranslational maturation process different from general mechanisms of metallocenter biosynthesis known so far: the unexpected behavior of a protein in a protein complex, which we named ''self-subunit swapping.'' metalloenzyme ͉ modification ͉ sulfinic acid ͉ sulfenic acid ͉ chaperone

OxdA shows an absorption spectrum with a Soret peak that is characteristic of heme, demonstrating that it is a hemoprotein. For its activity, this enzyme required a reducing reagent, Na 2 S 2 O 4 , but did not require FMN, which is crucial for the Bacillus enzyme. The enzymatic reaction was found to be catalyzed when the heme iron of the enzyme was in the ferrous state. Calcium as well as iron was included in the enzyme. OxdA reduced by Na 2 S 2 O 4 had a molecular mass of 76.2 kDa and consisted of two identical subunits. The kinetic parameters of OxdA indicated that aliphatic aldoximes are more effective substrates than aromatic aldoximes. A variety of spectral shifts in the absorption spectra of OxdA were observed upon the addition of each of various compounds (i.e. redox reagents and heme ligands). Moreover, the addition of the substrate to OxdA gave a peak that would be derived from the intermediate in the nitrile synthetic reaction. P. chlororaphis B23 grew and showed the OxdA activity when cultured in a medium containing aldoxime as the sole carbon and nitrogen source. Together with these findings, Western blotting analysis of the extracts using anti-OxdA antiserum revealed that OxdA is responsible for the metabolism of aldoxime in vivo in this strain.

Streptomycetes produce useful enzymes and a wide variety of secondary metabolites with potent biological activities (e.g., antibiotics, immunosuppressors, pesticides, etc.). Despite their importance in the pharmaceutical and agrochemical fields, there have been no reports for practical expression systems in streptomycetes. Here, we developed a ''PnitA-NitR'' system for regulatory gene expression in streptomycetes based on the expression mechanism of Rhodococcus rhodochrous J1 nitrilase, which is highly induced by an inexpensive and safe inducer, -caprolactam. Heterologous protein expression experiments demonstrated that the system allowed suppressed basal expression and hyper-inducible expression, yielding target protein levels of as high as Ϸ40% of all soluble protein. Furthermore, the system functioned in important streptomycete strains. Thus, the P nitA-NitR system should be a powerful tool for improving the productivity of various useful products in streptomycetes.

Isonitrile containing an NϵC triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K m value for cyclohexyl isocyanide was 16.2 mM. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.

The incorporation of cobalt into low molecular mass nitrile hydratase (L-NHase) of Rhodococcus rhodochrous J1 has been found to depend on the ␣-subunit exchange between cobalt-free L-NHase (apo-L-NHase lacking oxidized cysteine residues) and its cobalt-containing mediator (holo-NhlAE containing Cys-SO 2 ؊ and Cys-SO ؊ metal ligands), this novel mode of post-translational maturation having been named self-subunit swapping, and NhlE having been recognized as a self-subunit swapping chaperone (Zhou, Z., Hashimoto, Y., Shiraki, K., and Kobayashi, M. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 14849 -14854). We discovered here that cobalt was inserted into both the cobaltfree NhlAE (apo-NhlAE) and the cobalt-free ␣-subunit (apo-␣-subunit) in an NhlE-dependent manner in the presence of cobalt and dithiothreitol in vitro. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis revealed that the non-oxidized cysteine residues in apo-NhlAE were posttranslationally oxidized after cobalt insertion. These findings suggested that NhlE has two activities, i.e. cobalt insertion and cysteine oxidation. NhlE not only functions as a self-subunit swapping chaperone but also a metallochaperone that includes a redox function. Cobalt insertion and cysteine oxidation occurred under both aerobic and anaerobic conditions when Co 3؉ was used as a cobalt donor, suggesting that the oxygen atoms in the oxidized cysteines were derived from water molecules but not from dissolved oxygen. Additionally, we isolated apo-NhlAE after the self-subunit swapping event and found that it was recycled for cobalt transfer into L-NHase.

We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B 12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B 12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri-and tetra-methylated compounds that are not part of the vitamin B 12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B 12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.Uroporphyrinogen (urogen) III methyltransferase, a key enzyme in the biosynthetic pathways of vitamin B 12 and siroheme, catalyzes the S-adenosyl-L-methionine (SAM)-dependent bismethylation of its substrate, urogen III, resulting in the formation of dihydrosirohydrochlorin (known as precorrin-2 in the vitamin B 12 pathway). In the biosynthesis of vitamin B 12 in the anaerobe Propionibacterium freudenreichii, labeling experiments have indicated that cobalt is inserted soon after the formation of precorrin-2 (3, 20), and the recent isolation of a cobalt-containing tetramethylated corphinoid, possibly an intermediate, from this organism supports these observations (3). Cobalt insertion in the vitamin B 12 pathway of the aerobe Pseudomonas denitrificans, however, occurs at a much later stage with insertion into hydrogenobyrinic acid diamide (6).Urogen III methyltransferase has been purified to homogeneity from several different organisms, and the nucleotide sequences of the corresponding genes have been determined, revealing that the enzyme exists in at least two forms. One form, encoded by the cysG gene, is required for siroheme and, thus, for cysteine synthesis in Escherichia coli (14,31,35) and siroheme and vitamin B 12 synthesis in Salmonella typhimurium (8, 10). The ...

The complete nucleotide sequence of pRGO1, a cryptic plasmid from Propionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G؉C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4, orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containing orf1 (repA), orf2 (repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 ؋ 10 6 CFU/g of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 10 4 to 10 7 CFU/g of DNA. The vector was stably maintained in strains of P. freudenreichii subsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp. freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.Propionibacteria, which have a wide range of probiotic activity, are used in making dairy foods, such as cheese, for the production of vitamin B 12 , tetrapyrrole compounds, and propionic acid (8,15,21), in bread baking, as starters for ensilage, and in some pharmaceutical preparations (35). To elucidate the biosynthetic pathways of vitamin B 12 and siroheme in Propionibacterium, we previously identified several genes coding for the enzymes involved in production of tetrapyrrole derivatives (hemYHBXRL) (11,12) and vitamin B 12 (cobA, cbiO) (30).Development of genetic manipulation in propionibacteria has progressed slowly due to a lack of detailed information on the genetics of the bacteria and a lack of an appropriate plasmid that can serve as a possible transformation vector. A number of plasmids from Propionibacterium acidipropionici, P. freudenreichii, and P. jensenii, ranging in size from 4.4 to more than 119 MDa, have been described (19,24). However, neither analysis of a plasmid DNA sequence nor construction of a vector for propionibacteria has been reported. To establish a versatile vector system to facilitate genetic analysis and to allow the transfer of a gene of interest, we investigated the development of a host-vector system in propionibacteria.We succeeded in determining the co...