We evaluated the effect of an oral administration of a plant-derived lactic acid bacterium, Pediococcus pentosaceus LP28 (LP28), on metabolic syndrome by using high fat diet-induced obese mice. The obese mice were divided into 2 groups and fed either a high fat or regular diet for 8 weeks. Each group was further divided into 3 groups, which took LP28, another plant-derived Lactobacillus plantarum SN13T (SN13T) or no lactic acid bacteria (LAB). The lean control mice were fed a regular diet without inducing obesity prior to the experiment. LP28 reduced body weight gain and liver lipid contents (triglyceride and cholesterol), in mice fed a high fat diet for 8 weeks (40%, 54%, and 70% less than those of the control group without LAB, and P = 0.018, P<0.001, and P = 0.021, respectively), whereas SN13T and the heat treated LP28 at 121°C for 15 min were ineffective. Abdominal visceral fat in the high fat diet mice fed with LP28 was also lower than that without LAB by 44%, although it was not significant but borderline (P = 0.076). The sizes of the adipocytes and the lipid droplets in the livers were obviously decreased. A real-time PCR analyses showed that lipid metabolism-related genes, such as CD36 (P = 0.013), SCD1 encoding stearoyl-CoA desaturase 1 (not significant but borderline, P = 0.066), and PPARγ encoding peroxisome proliferator-activated receptor gamma (P = 0.039), were down-regulated by taking LP28 continuously, when compared with those of the control group. In conclusion, LP28 may be a useful LAB strain for the prevention and reduction of the metabolic syndrome.

Tyrosinase (EC 1.14.18.1), a copper-containing monooxygenase, catalyzes the conversion of phenol to the corresponding ortho-quinone. The Streptomyces tyrosinase is generated as a complex with a “caddie” protein that facilitates the transport of two copper ions into the active center. In our previous study, the Tyr⁹⁸ residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, has been found to be converted to a reactive quinone through the formations of the μ-η²:η²-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now—despite extensive studies for the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight models, and computer simulations—the catalytic mechanism has been unable to be made clear at an atomic level. To make the catalytic mechanism of tyrosinase clear, in the present study, the cryo-trapped crystal structures were determined at very high resolutions (1.16–1.70 Å). The structures suggest the existence of an important step for the tyrosinase reaction that has not yet been found: that is, the hydroxylation reaction is triggered by the movement of Cu^A, which induces the syn-to-anti rearrangement of the copper ligands after the formation of μ-η²:η²-peroxo-dicopper(II) core. By the rearrangement, the hydroxyl group of the substrate is placed in an equatorial position, allowing the electrophilic attack to the aromatic ring by the Cu₂O₂ oxidant. Tyrosinase is an enzyme that controls a rate-limiting reaction of melanogenesis: it catalyzes the conversion of a phenol to the corresponding ortho-quinone. Streptomyces tyrosinase is formed as a complex, with a “caddie” protein that assists with the transport of the two copper ions into the enzyme’s active center. In our previous study, we showed that the Tyr⁹⁸ residue in the caddie protein, which is accommodated in the pocket of active center of tyrosinase, is converted to a reactive quinone through the formations of the μ-η²:η²-peroxo-dicopper(II) and Cu(II)-dopasemiquinone intermediates. Until now—despite extensive studies of the tyrosinase reaction based on the crystallographic analysis, low-molecular-weight model systems, and computer simulations—the catalytic mechanism was unclear at an atomic level. To understand the catalytic mechanism of tyrosinase in detail, we determined the cryo-trapped crystal structures at very high resolutions, which suggest an important new step for the tyrosinase reaction: the hydroxylation reaction triggered by the movement of Cu^A, which induces the syn-to-anti rearrangement of the copper ligands after the formation of μ-η²:η²-peroxo-dicopper(II) core.

The Cu(II)-soaked crystal structure of tyrosinase that is present in a complex with a protein, designated “caddie,” which we previously determined, possesses two copper ions at its catalytic center. We had identified two copper-binding sites in the caddie protein and speculated that copper bound to caddie may be transported to the tyrosinase catalytic center. In our present study, at a 1.16–1.58 Å resolution, we determined the crystal structures of tyrosinase complexed with caddie prepared by altering the soaking time of the copper ion and the structures of tyrosinase complexed with different caddie mutants that display little or no capacity to activate tyrosinase. Based on these structures, we propose a molecular mechanism by which two copper ions are transported to the tyrosinase catalytic center with the assistance of caddie acting as a metallochaperone.

Bleomycin (Bm) in the culture broth ofStreptomyces verticillus is complexed with Cu²⁺(Cu(II)). In the present study, we determined the x-ray crystal structures of the Cu(II)-bound and the metal-free types of Bm at a high resolution of 1.6 and 1.8 Å, respectively, which are complexed with a Bm resistance determinant from Bm-producing S. verticillus, designated BLMA. In the current model of Cu(II)·Bm complexed with BLMA, two Cu(II)·Bm molecules bind to the BLMA dimer. The electron density map shows that the copper ion is clearly defined in the metal-binding domain of the Bm molecule. The metal ion is penta-coordinated by a tetragonal monopyramidal cage of nitrogens and binds to the primary amine of the β-aminoalanine moiety of Bm. The binding experiment between Bm and BLMA showed that each of the two Bm-binding pockets has a different dissociation constant (K _{d 1} andK _{d 2}). TheK _{d 1} value of 630 nm for the first Bm binding is larger than the K _{d 2}value of 120 nm, indicating that the first Bm binding gives rise to a cooperative binding of the second Bm to the other pocket.

We have established a novel assay method based on circular dichroism that can be used for the kinetic study of the activity of amino acid racemases, such as ALR (alanine racemase). Although an enzyme-coupled assay method has been used to measure racemase activity, the CD method is superior to the enzyme assay because it can accurately determine the immediate changes of an enantiomer on racemization between its L- and D-forms. The enzyme-coupled assay requires D-amino acid oxidase, which is inactivated by an inhibitor of ALR, D-cycloserine. This indicates that the inhibitory kinetic study for ALR with D-cycloserine by the enzyme-coupled assay method is restricted to the analysis of only the reaction resulting in the formation of L-Ala from D-Ala. However, since the CD assay does not require the coupled enzyme, it can be used to comprehensively evaluate the reactions that result in the formation both of D-Ala from L-Ala and of L-Ala from D-Ala at several substrate concentrations. Streptomyces ALR also catalyses the formation of D-Ser from L-Ser and of L-Ser from D-Ser, but the catalytic constants (k_cat) are 4- and 10-fold lower than those for the formation of D-Ala from L-Ala and of L-Ala from D-Ala respectively.

An antibiotic, d-cycloserine (DCS), inhibits the catalytic activities of alanine racemase (ALR) and d-alanyl-d-alanine ligase (DDL), which are necessary for the biosynthesis of the bacterial cell wall. In this study, we cloned both genes encoding ALR and DDL, designated alrS and ddlS, respectively, from DCS-producing Streptomyces lavendulae ATCC25233. Each gene product was purified to homogeneity and characterized. Escherichia coli, transformed with a pET vector carrying alrS or ddlS, displays higher resistance to DCS than the same host carrying the E. coli ALR- or DDL-encoded gene inserted into the pET vector. Although the S. lavendulae DDL was competitively inhibited by DCS, the K_i value (920 μm) was obviously higher (40∼100-fold) than those for E. coli DdlA (9 μm) or DdlB (27 μm). The high K_i value of the S. lavendulae DDL suggests that the enzyme may be a self-resistance determinant in the DCS-producing microorganism. Kinetic studies for the S. lavendulae ALR suggest that the time-dependent inactivation rate of the enzyme by DCS is absolutely slower than that of the E. coli ALR. We conclude that ALR from DCS-producing S. lavendulae is also one of the self-resistance determinants.