Kenichi Uchida

Metabolite Profiling of Hydroxycinnamate Derivatives in Plasma and Urine after the Ingestion of Coffee by Humans: Identification of Biomarkers of Coffee Consumption

Human subjects drank coffee containing 412 μmol of chlorogenic acids, and plasma and urine were collected 0 to 24 h after ingestion and were analyzed by high-performance liquid chromatography-mass spectrometry. Within 1 h, some of the components in the coffee reached nanomole peak plasma concentrations (Cmax), whereas chlorogenic acid metabolites, including caffeic acid-3-O-sulfate and ferulic acid-4-O-sulfate and sulfates of 3- and 4-caffeoylquinic acid lactones, had higher Cmax values. The short time to reach Cmax (Tmax) indicates absorption of these compounds in the small intestine. In contrast, dihydroferulic acid, its 4-O-sulfate, and dihydrocaffeic acid-3-O-sulfate exhibited much higher Cmax values (145–385 nM) with Tmax values in excess of 4 h, indicating absorption in the large intestine and the probable involvement of catabolism by colonic bacteria. These three compounds, along with ferulic acid-4-O-sulfate and dihydroferulic acid-4-O-glucuronide, were also major components to be excreted in urine (8.4–37.1 μmol) after coffee intake. Feruloylglycine, which is not detected in plasma, was also a major urinary component (20.7 μmol excreted). Other compounds, not accumulating in plasma but excreted in smaller quantities, included the 3-O-sulfate and 3-O-glucuronide of isoferulic acid, dihydro(iso)ferulic acid-3-O-glucuronide, and dihydrocaffeic acid-3-O-glucuronide. Overall, the 119.9 μmol excretion of the chlorogenic acid metabolites corresponded to 29.1% of intake, indicating that as well as being subject to extensive metabolism, chlorogenic acids in coffee are well absorbed. Pathways for the formation of the various metabolites within the body are proposed. Urinary dihydrocaffeic acid-3-O-sulfate and feruloylglycine are potentially very sensitive biomarkers for the consumption of relatively small amounts of coffee.

Confirming Stereochemical Structures of Strigolactones Produced by Rice and Tobacco

Summary Major strigolactones produced by rice (Oryza sativa L.) and tobacco (Nicotiana tabacum L.) were purified and their stereochemical structures were determined definitely by comparing with optically pure synthetic standards for spectroscopic data.

Carboxyl proteinase from Pseudomonas defines a novel family of subtilisin-like enzymes.

The crystal structure of a pepstatin-insensitive carboxyl proteinase from Pseudomonas sp. 101 (PSCP) has been solved by single-wavelength anomalous diffraction using the absorption peak of bromide anions. Structures of the uninhibited enzyme and of complexes with an inhibitor that was either covalently or noncovalently bound were refined at 1.0–1.4 Å resolution. The structure of PSCP comprises a single compact domain with a diameter of ∼55 Å, consisting of a seven-stranded parallel β-sheet flanked on both sides by a number of helices. The fold of PSCP is a superset of the subtilisin fold, and the covalently bound inhibitor is linked to the enzyme through a serine residue. Thus, the structure of PSCP defines a novel family of serine-carboxyl proteinases (defined as MEROPS S53) with a unique catalytic triad consisting of Glu 80, Asp 84 and Ser 287.

Emission Mechanism of Floral Scent in Petunia axillaris

The mechanism of floral scent emission was studied in Petunia axillaris, a plant with a diurnal rhythm of scent output. The emission rate of each volatile compound oscillated in synchrony with its endogenous concentration, so that the intensity of the floral scent appeared to be determined by the endogenous concentrations. The composition of major volatiles in the flower tissue and the flower headspace showed characteristic differences. A negative correlation was found between the boiling points of the volatile compounds and the ratio of their emitted and endogenous concentrations, indicating that the composition of the floral scent depends directly on the endogenous composition of the volatile compounds. We conclude that in P. axillaris, the physiological regulation of floral scent emission operates not in the vaporization process but in the control of the endogenous concentrations of volatiles through biosynthesis and metabolic conversion.

The 1.4 Å Crystal Structure of Kumamolysin: A Thermostable Serine-Carboxyl-Type Proteinase

Kumamolysin is a thermostable endopeptidase from Bacillus novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 A resolution. As its Pseudomonas homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.

Three-dimensional structure determination of a uniformly labeled molecule by frequency-selective dipolar recoupling under magic-angle spinning

The complete three-dimensional (3D) structure of a glycylisoleucine (Gly-Ile) molecule was determined by individually measuring six dihedral angles with a frequency-selective homonuclear dipolar recoupling method, R2TR (rotational resonance in the tilted rotating frame), using a powder sample of diluted uniformly 13,15-labeled Gly-Ile. Each dihedral angle was obtained by recoupling a dipolar interaction between three or four bonds distant spins concerned or observing a dipolar correlation 2D powder pattern. The 3D structure of a Gly-Ile molecule was also determined by X-ray crystallography, and a good agreement with the NMR result was obtained. The results demonstrate that the R2TR method in a uniformly labeled powder sample can provide the 3D structure without the need to prepare a lot of selectively labeled samples.

Avenaol, a germination stimulant for root parasitic plants from Avena strigosa

Root exudates from the allelopathic plant, black oat (Avena strigosa Schreb.), were found to contain at least six different germination stimulants for root parasitic plants, but no known strigolactones (SLs). One of these germination stimulants was purified and named avenaol. Its HR-ESI-TOFMS analysis indicated that the molecular formula of avenaol is C20H24O7, and thus it contains an additional carbon compared with known C19-SLs. Its structure was determined as 5-((E)-(5-(3-hydroxy-1,5,5-trimethyl-2-oxobicyclo[4.1.0]heptan-7-yl)-2-oxodihydrofuran-3(2H)-ylidene)methoxy)-3-methylfuran-2(5H)-one, by 1D and 2D NMR spectroscopy, and ESI- and EI-MS spectrometry. Although avenaol contains the C-D moiety, the common structural feature for all known SLs, it lacks the B ring and has an additional carbon atom between the A and C rings. Avenaol is a potent germination stimulant of Phelipanche ramosa seeds, but only a weak stimulant for seeds of Striga hermonthica and Orobanche minor.

Biosynthesis of lactacystin. Origin of the carbons and stereospecific NMR assignment of the two diastereotopic methyl groups

Abstract Biosynthetic pathway of lactacystin and its stereochemical aspect were investigated by feeding experiments of 13 C enriched compounds.

Biosynthesis of Quinolactacin A, a TNF Production Inhibitor

Quinolactacins, which inhibit tumor necrosis factor production, contain a quinolone skeleton conjugated with a γ-lactam. The biosynthesis of quinolactacin was investigated by feeding experiments using 13C single-labeled precursors (sodium [1-13C]acetate, DL-[1-13C;[shy;isoleucine, L-[methyl-13C]methionine, and sodium [1-13C]anthranilate) and D-[U-13C]glucose.

Methyl zealactonoate, a novel germination stimulant for root parasitic weeds produced by maize

One of the germination stimulants for root parasitic weeds produced by maize (Zea mays) was isolated and named methyl zealactonoate (1). Its structure was determined to be methyl (2E,3E)-4-((RS)-3,3-dimethyl-2-(3-methylbut-2-en-2-yl)-5-oxotetrahydrofuran-2-yl)-2-((((R)-4-methyl-5-oxo-2,5-dihydrofran-2-yl)oxy)methylene)but-3-enoate using by 1D and 2D NMR spectroscopy and ESI and EI-MS spectrometry. Feeding experiments with 13C-carlactone (CL), a biosynthetic intermediate for strigolactones, confirmed that 1 is produced from CL in maize. Methyl zealactonoate strongly elicits Striga hermonthica and Phelipanche ramosa seed germination, while Orobanche minor seeds are 100-fold less sensitive to this stimulant.