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Geobacillus kaustophilus is a thermophilic bacterium that grows at temperatures ranging between 42 and 74 °C. Here, we modified this organism to produce the thermolabile protein (PyrFA) or its thermostable variant (PyrFV) and analyzed the transcriptome and growth efficiency profiles of the resultant strains. In the producer of PyrFA, the transcriptome profile was changed to facilitate ATP synthesis from NADH without pooling reduced quinones. This change implies that PyrFA production at elevated temperatures places an energy burden on cells potentially to maintain protein homeostasis. This was consistent with the observation that the PyrFA producer grew slower than the PyrFV producer at > 45 °C and had a lower cellular fitness. Similar growth profiles were also observed in the PyrFA and PyrFV producers derived from another thermophile (Geobacillus thermodenitrificans) but not in those from Escherichia coli at 30 °C. Thus, we suggest that the production of thermolabile proteins impairs host survival at higher temperatures; therefore, thermophiles are under evolutionary selection for thermostable proteins regardless of whether their functions are associated with survival advantages. This hypothesis provides new insights into evolutionary protein selection in thermophiles and suggests an engineering approach to select thermostable protein variants generated via random gene mutagenesis.

Fucoidan is a hetero-sulfated polysaccharide found in brown algae and has received much attention as an ingredient in functional and health foods. The marine bacterial strain Luteolibacter algae H18 degrades fucoidan from Cladosiphon okamuranus. We purified the fucoidanase from a cell-free extract of L. algae H18, used it to decrease the molecular weight of deacetylated-fucoidan, determined the N-terminal amino acid sequence of the enzyme, and identified the gene involved in the degradation of fucoidan, fct114, in a draft genome sequence of strain H18. The gene product was heterologously produced in Escherichia coli and demonstrated to catalyze the degradation of deacetylated-fucoidan into lower molecular weight fragments. The mass of the gene product Fct114 is 112 kDa (1026 amino acid residues). The general properties of the enzyme were investigated by measuring the amount of reducing ends produced from deacetylated-fucoidan during the reaction. The enzyme was inactive toward fucoidans from other brown seaweed species or toward polysaccharides such as alginic acid, carrageenan, hyaluronic acid, and chondroitin sulfate. The amino acid sequence of Fct114 shared less than 25% identity and had no conserved motifs when compared with previously identified fucoidanases from other marine bacterial strains. These data suggest that Fct114 is a novel polysaccharide-degrading enzyme.

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.

Geobacillus spp. are moderate thermophiles that have great potential for use in diverse applications. For effective utilization of the species, genetic tools have been extensively studied; however, an overexpression vector remains to be developed. Here we constructed a plasmid vector that can shuttle between Escherichia coli and Geobacillus spp., and which contained a maltose-inducible promoter from Geobacillus kaustophilus HTA426. Although the vector (termed pGKE119) was originally designed for basal gene expression, it surprisingly directed robust protein production in G. kaustophilus. Protein production essentially occurred in an auto-inducible manner without maltose; however, some proteins were produced more efficiently in the presence of maltose. Although the productivity was affected by culture conditions, three proteins were successfully produced with abundance ratios of 12–27% (on a total protein basis) and yields of 77–170 mg (per L culture). pGKE119 directed substantial protein production even in Geobacillus subterraneus, Geobacillus thermoglucosidasius, and Geobacillus thermoleovorans. This suggests that pGKE119 can use a range of Geobacillus spp. as hosts and widely expand their genetic toolbox. Because Geobacillus spp. are highly proliferative bacteria that are distinct from organisms used as protein production hosts, pGKE119 may also provide a novel platform for hyperproduction of recombinant proteins.

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

Azoreductases reductively cleave azo linkages by using NAD(P)H as an electron donor. The enzymes are widely found in bacteria and act on numerous azo dyes, which allow various unique applications. This review describes primary amino acid sequences, structures, substrates, physiological roles, and biotechnological applications of bacterial azoreductases to discuss their remarkable diversification. According to primary sequences, azoreductases were classified phylogenetically into four main clades. Most members of clades I-III are flavoproteins, whereas clade IV members include flavin-free azoreductases. Clades I and II prefer NADPH and NADH, respectively, as electron donors, whereas other members generally use both. Several enzymes formed no clades; moreover, some bacteria produce azoreductases with longer primary structures than those hitherto identified, which implies further diversification of bacterial azoreductases. The crystal structures commonly reveal the Rossmann folds; however, ternary structures are moderately varied with different quaternary conformation. Although physiological roles are obscure, several azoreductases have been shown to act on metabolites such as flavins, quinones, and metal ions more efficiently than on azo dyes. Considering that many homologs exclusively act on these metabolites, it is possible that azoreductases are actually side activities of versatile reductases that act on various substrates with different specificities. In parallel, this idea raises the possibility that homologous enzymes, even if these are already defined as other types of reductases, widely harbor azoreductase activities. Although azoreductases for which their genes have been identified are not abundant, it may be simple to identify azoreductases of biotechnological importance that have novel substrate specificities.

Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsrH258Y functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsrH258Y was more thermostable than that from tsr. In fact, the tsrH258Y gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

Enhancing the thermostability of thermolabile enzymes extends their practical utility. We previously demonstrated that an error-prone thermophile derived from Geobacillus kaustophilus HTA426 can generate mutant genes encoding enzyme variants that are more thermostable than the parent enzyme. Here, we used this approach, termed as thermoadaptation-directed enzyme evolution, to increase the thermostability of the chloramphenicol acetyltransferase (CAT) of Staphylococcus aureus and successfully generated a CAT variant with an A138T replacement (CAT(A138T)). This variant was heterologously produced, and its enzymatic properties were compared with those of the wild type. We found that CAT(A138T) had substantially higher thermostability than CAT but had comparable activities, showing that the A138T replacement enhanced protein thermostability without affecting the catalytic activity. Because variants CAT(A138S) and CAT(A138V), which were generated via in vitro site-directed mutagenesis, were more thermostable than CAT, the thermostability enhancement resulting from the A138T replacement can be attributed to both the presence of a hydroxyl group and the bulk of the threonine side chain. CAT(A138T) conferred chloramphenicol resistance to G. kaustophilus cells at high temperature more efficiently than CAT. Therefore, the gene encoding CAT(A138T) may be useful as a genetic marker in Geobacillus spp. Notably, CAT(A138T) generation was achieved only by implementing improved procedures (plasmid-based mutations on solid media); previous procedures (chromosome-based mutations in liquid media) were unsuccessful. This result suggests that this improved procedure is crucial for successful thermoadaptation-directed evolution in certain cases and increases the opportunities for generating thermostable enzymes.

Fucoidan is an α-l-fucopyranosyl polymer found in seaweeds with forms that have acetyl and sulfuric modifications and derivatives that are lower and/or diversified, with modifications that have attracted interest as potential bioactive substances. We identified the gene for a fucoidan deacetylase that cleaves acetyl moieties from fucoidan and thereby contributes to fucoidan utilization in the marine bacterium Luteolibacter algae H18. Fucoidan deacetylase was purified to homogeneity from a cell-free extract of L. algae H18, and used to determine the internal amino acid sequence and identify the gene, fud, in a draft genome sequence of the H18 strain. The gene product was heterologously produced in Escherichia coli and was demonstrated to catalyze fucoidan deacetylation, but not desulfation, and degradation into lower forms. In addition to fucoidan deacetylation, the enzyme catalyzed the hydrolysis of p-nitrophenyl esters with organic acids, and p-nitrophenyl acetate was the best substrate among those tested. The present study provides a new tool for fucoidan degradation, potentially expanding investigations on fucoidan derivatives.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

Genipin, a herbal iridoid, is known to have both neuroprotective and neuritogenic activity in neuronal cell lines. As it is structurally similar to tetrahydrobiopterin, its activity is believed to be nitric oxide (NO)-dependent. We previously proposed a novel neuroprotective activity of a genipin derivative, (1R)-isoPropyloxygenipin (IPRG001), whereby it reduces oxidative stress in RGC-5, a neuronal precursor cell line of retinal origin through protein S-nitrosylation. In the present study, we investigated another neuritogenic property of IPRG001 in RGC-5 cells and retinal explant culture where in we focused on the NO-cGMP-dependent and protein S-nitrosylation pathways. IPRG001 stimulated neurite outgrowth in RGC-5 cells and retinal explant culture through NO-dependent signaling, but not NO-dependent cGMP signaling. Neurite outgrowth with IPRG001 requires retinoic acid receptor β (RARβ) expression, which is suppressed by an RAR blocking agent and siRNA inhibition. Thereby, we hypothesized that RARβ expression is mediated by protein S-nitrosylation. S-nitrosylation of histone deacetylase 2 is a key mechanism in chromatin remodeling leading to transcriptional gene activation. We found a parallelism between S-nitrosylation of histone diacetylase 2 and the induction of RARβ expression with IPRG001 treatment. The both neuroprotective and neuritogenic activities of genipin could be a new target for the regeneration of retinal ganglion cells after glaucomatous conditions.

Introduction: IgM composes a part of the B cell receptor, and its expression is restricted to B cell lineage cells. There have been several reports that refractory B cell malignant cells frequently express IgM. For example, activated B cell-like (ABC) diffuse large cell lymphoma (DLBCL) is a subtype with a poor prognosis following R-CHOP treatment (Lenz et al., N Engl J Med, 2008), and the tumor cells generally express IgM-type immunoglobulin (Ruminy et al., Leukemia, 2011). In addition, IgM-expressing chronic lymphocytic leukemia (CLL) was also found to have a poor prognosis (D'Avola et al., Blood, 2016). Therefore, IgM is a promising therapeutic target for refractory B cell malignancies. However, anti-IgM antibodies may not function fully in the body due to the presence of soluble IgM. To improve the selectivity to the IgM expressing cells, we produced a bispecific antibody bound to IgM and HLA-DR, which are both expressed by B cells, and it was able to bind IgM+HLA-DR+ cells in the presence of soluble IgM. Thus, the aim of the present study was to examine the in vitro anti-tumor activity of the anti-IgM/HLA-DR bispecific antibody against B cell lymphoma, and to clarify its efficacy and tolerability in non-human primates (NHP). Methods: BTA106 is the bispecific antibody that can bind to IgM and HLA-DR, and BTA111 is glycoengineered BTA106. To investigate their direct inhibitory effects and Fc-mediated effects against B cell lymphoma cell lines, a growth inhibition assay, antibody-dependent cell cytotoxicity (ADCC) assay and complement dependent cytotoxicity (CDC) assay were performed. In the growth inhibition assay and CDC assay, cell proliferation was measured by WST-8. In the ADCC assay, human peripheral blood mononuclear cells were used as effector cells. Dead cells were stained with propidium iodide and measured by flow cytometry. JeKo-1 (Mantle cell lymphoma, MCL), Ramos (Burkitt's lymphoma, BL), RL (DLBCL), B104 (B cell lymphoma) and two rituximab-resistant cell lines, RL-4RH (Czuczman et al., Clin Cancer Res., 2008) and RRBL1 (generously provided by Dr. Tomita, Tomita et al., Int J Hematol., 2007), were used. To clarify the efficacy and tolerability of BTA106 in NHP, a dose escalation study and single-dose administration study were performed using cynomolgus monkeys. Results: BTA106 and BTA111 demonstrated direct growth inhibition activity, and CDC and ADCC activity, although their intensities differed among cell lines. In the case of rituximab-resistant cell lines, RRBL1 and RL-4RH, BTA106 and BTA111 exhibited weak or no growth inhibitory effects, but their CDC and ADCC activity was superior to that of rituximab. Development of resistance to rituximab is also a poor prognostic factor and remains problematic. Thus, BTA106 and BTA111 are expected as therapeutic antibodies to overcome rituximab resistance. A dose escalation study and single dose study were performed in cynomolgus monkeys. BTA106 greatly reduced peripheral B cells. Moreover, after the administration of BTA106, lymph follicles disappeared in axillary lymph nodes. In these studies, decreased locomotor activity and vomiting were observed; however, these symptoms resolved 2 hrs after injection. Other BTA106-related clinical toxicities or changes in body weight were not observed. Conclusions: In the in vitro studies, BTA106 and BTA111 demonstrated significant anti-tumor effects on rituximab-sensitive and -resistant B cell lymphoma cell lines. The efficacy and toxicity studies in cynomolgus monkeys revealed that BTA106 depletes peripheral and lymph node B cells, and it was well tolerated. BTA111 was considered to be significantly more effective than BTA106 due to glycoengineering, which induced high ADCC activity. These data suggest that BTA111 is an effective and potent therapeutic antibody for B cell malignancies, especially rituximab-resistant B cell lymphoma. Currently, a repeated administration study of BTA111 with cynomolgus monkeys to evaluate its efficacy and toxicity is ongoing. Disclosures Ohashi: Zenyaku Kogyo Co., Ltd.: Employment. Miyashita:Zenyaku Kogyo Co., Ltd.: Employment. Nagata:Zenyaku Kogyo Co., Ltd.: Employment. Tatebe:Zenyaku Kogyo Co., Ltd.: Employment. Otsuka:Zenyaku Kogyo Co., Ltd.: Employment. Suzuki:Zenyaku Kogyo Co., Ltd.: Employment. Sasaki:Zenyaku Kogyo Co., Ltd.: Employment. Enami:Zenyaku Kogyo Co., Ltd.: Employment. Tsukada:Zenyaku Kogyo Co., Ltd.: Employment. Fukushima:Zenyaku Kogyo Co., Ltd.: Employment.

o-Ethynylbenzyl phenyl selenides regioselectively reacted with trifluoromethanesulfonic acid to afford the (Z)-1-methylidene-2-phenyl-1,3-dihydro-1H-benzo[c]selenophenium salts as the major products during the 5-exo-dig mode cyclization in good yields together with minor E isomers. The structure of the major (Z)-selenophenium salt was established by the single crystal X-ray crystallographic analysis using a tert-butyl derivative.

Previously, we reported that genipin, a herbal iridoid, had neuritogenic and neuroprotective actions on PC12 cells. Although nitric oxide (NO)-activated signalings were proposed to be neuritogenic, the neuroprotective action of genipin remains to be elucidated. From the standpoint of NO activation, we tested a possible protective mechanism through the nitrosative Kelch-like ECH-associated protein (Keap1)/NF-E2-related factor 2 (Nrf2)-antioxidant response element pathway in rat retinal ganglion cells (RGC-5 cells) in culture, and in vivo, against hydrogen peroxide and optic nerve injury (ONI), respectively, using a long-acting (1R)-isoPropyloxygenipin (IPRG001). IPRG001 induced NO generation and the expressions of antioxidative enzymes, such as heme oxygenase-1 (HO-1), in RGC-5 cells. The protective action of IPRG001 depended on HO-1 and NO induction. We found that S-nitrosylation of Keap1 by IPRG001 may contribute to translocation of Nrf2 to the nucleus and triggered transcriptional activation of antioxidative enzymes. Furthermore, apoptotic cells were increased and 4-hydroxy-2-nonenal was accumulated in rat retina following ONI. Pre-treatment with IPRG001 almost completely suppressed apoptosis and accumulation of 4-hydroxy-2-nonenal in RGCs following ONI accompanied by HO-1 induction. These data demonstrate for the first time that IPRG001 exerts neuroprotective action in RGCs in vitro and in vivo, through the Nrf2/antioxidant response element pathway by S-nitrosylation against oxidative stress.

The intramolecular cyclizations of four types of dibenzyl chalcogenols, which contained one or two ethynyl groups, were carried out. Either the double 5-exo or 6-endo-dig mode ring closure reaction regio- and stereoselectively proceeded to give the corresponding symmetrical bis(benzo[c]chalcogenophene) or bis(isochromene) derivatives from the dibenzyl chalcogenols having a diyne moiety. In contrast, the regioselective tandem 5-exo-dig mode intramolecular ring closure reactions of dibenzyl thiol and selenol having a mono-yne into a triple bond gave the trans-biisobenzothiophene and selenophene as the sole product. However, the similar cyclization of dibenzyl tellurol proceeded in both tandem 5-exo and 6-endo-dig modes to afford the trans-biisobenzotellurophene and ditellurachrysene in good yields with the ratio of 1:1. The X-ray structural analysis of these novel compounds is also presented.

We designed 1-alkyloxygenipins with the aim of improving the stability of genipins based on the structural and electronic properties of genipins, and prepared 1-alkyloxygenipins and examined their neuritogenic activities in PC12h cells. All genipin-derivatives exhibited electronic properties similar to those of genipin and induced significant neurite outgrowth. These compounds will be classified as nitric oxide synthase (NOS) activators (neuritogenic active compounds) since their lowest unoccupied molecular orbital (LUMO)-energies are similar to that of tetrahydrobiopterin (H4B). (1R)-isoPropyloxygenipin showed activity comparable to that of genipin, and unlike the parent compound genipin, it was found to be physiologically stable in rat liver homogenate.

Fumitremorgin C, a diketopiperazine mycotoxin produced by Aspergillus fumigatus, is a potent and specific inhibitor of breast cancer resistance protein (BCRP). Elucidation of the fumitremorgin C biosynthetic pathway provides a strategy for new drug design. A structure-activity relationship study based on metabolites related to the ftm gene cluster revealed that the process most crucial for inhibitory activity against BCRP was cyclization to form fumitremorgin C. To determine the gene involved in the cyclization reaction, targeted gene inactivation was performed with candidate genes in the ftm cluster. Analysis of the gene disruptants allowed us to identify ftmE, one of the cytochrome P450 genes in the cluster, as the gene responsible for the key step in fumitremorgin biosynthesis. Additionally, we demonstrated that the other two cytochrome P450 genes, ftmC and ftmG, were involved in hydroxylation of the indole ring and successive hydroxylation of fumitremorgin C, respectively.

The linear chromosome of Streptomyces griseus IFO13350 contains not only atypical telomere sequences but also probable pseudogenes for two typical telomeric proteins. Two identical operons (SGR98t-SGR97t near one telomere and SGR7041t-SGR7042t near the other telomere) in the terminal inverted repeat sequence were predicted to encode a novel pair of telomeric proteins. SGR97t, a 185-amino-acid protein showing only 18% amino acid sequence identity to typical terminal proteins of Streptomyces, was found to be attached to the chromosomal ends, as determined by immunological analysis. On the other hand, electrophoretic mobility shift assays showed that SGR98t, an 837-amino-acid protein having a DnaB-like helicase C-terminal domain, was capable of binding specifically to the single-stranded terminal DNA corresponding to the 3' overhang of the replication intermediate. These results indicate that SGR97t (and SGR7042t) and SGR98t (and SGR7041t) were the functional telomeric proteins in the replication of the linear chromosome of S. griseus IFO13350.

A cDNA from soybean (Glycine max (L.) Merr.), GmIF7MaT, encoding malonyl-CoA:isoflavone 7-O-glucoside-6''-O-malonyltransferase, was cloned and characterized. Soybeans produce large amounts of isoflavones, which primarily accumulate in the form of their 7-O-(6''-O-malonyl-beta-D-glucosides). The cDNA was obtained by a homology-based strategy for the cDNA cloning of some flavonoid glucoside-specific malonyltransferases of the BAHD family. The expressed gene product, GmIF7MaT, efficiently catalyzed specific malonyl transfer reactions from malonyl-CoA to isoflavone 7-O-beta-D-glucosides yielding the corresponding isoflavone 7-O-(6''-O-malonyl-beta-D-glucosides) (IF7MaT activity). The k(cat) values of GmIF7MaT were much greater than those of other flavonoid glucoside-specific malonyltransferases with their preferred substrates, while the K(m) values were at comparable levels. GmIF7MaT was expressed in the roots of G. max seedlings more abundantly than in hypocotyl and cotyledon. Native IF7MaT activity was also observed in the roots, suggesting that GmIF7MaT is involved in the biosynthesis from isoflavone 7-O-beta-D-glucosides to the corresponding isoflavone 7-O-(6''-O-malonyl-beta-D-glucosides) in G. max. This protein is a member of flavonoid glucoside-specific acyltransferases in the BAHD family.

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

The present investigation was undertaken to compare the structural and electronic properties of genipin with those of tetrahydrobiopterin (H4B), an essential cofactor in neuronal nitric oxide synthase (nNOS), and 4-aminotetrahydrobiopterin (4-amino-H4B), an inhibitor of nNOS, using computer-assisted molecular modeling techniques. Molecular modeling, superimposing, and docking simulation, in addition to LUMO-energy calculation, revealed that genipin has structural and electronic properties that markedly resemble those of H4B.

Some polyacylated anthocyanins, anthocyanins containing two or more aromatic acyl groups, are blue in color within a wide range of pH values, including acidic and neutral conditions even in the absence of any co-pigments or metal ions. We engineered a mutant of a malonyl-CoA:anthocyanin 5-O-glucoside-6� -O-malonyltransferase of Salvia splendens (Ss5MaT1), Ss5AT306, which has an acquired specificity for hydroxycinnamoyl-CoA and is able to produce novel polyacylated anthocyanins. Ss5AT306 showed 6� -O-hydroxycinnamoyltransferase activity toward some anthocyanins in addition to the 6� -O-malonyltransferase activity retained (relative activities for acyl transfer to shisonin: malonyl-CoA, 100%; p-coumaroyl-CoA, 132%; caffeoyl-CoA, 103%). This alteration of acyl-donor specificity was achieved by the substitutions of only three contiguous amino acid residues, Val39-Arg40-Arg41, to the corresponding residues of anthocyanin aromatic acyltransferases, Met-Leu-Gln, suggesting that these amino acid residues are key residues governing the malonyl-CoA specificity of Ss5MaT1. Through the use of Ss5AT306, a novel polyacylated anthocyanin, p- coumaroylshisonin, with one aromatic acyl group in each of the 3-O-glucosyl and 5-O-glucosyl moieties of its structure, was produced and characterized in vitro. p-Coumaroylshisonin was bluer in color and displayed stronger color intensity than did shisonin, implying that accumulation of polyacylated anthocyanins such as p-coumaroylshisonin cause the modulation of flower colors.

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 ''-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.

This report describes efficient plasmid uptake by the thermophile Geobacillus kaustophilus HTA426 by means of a ternary conjugation system, which was used to construct thermophile DNA libraries for G. kaustophilus and to identify the genes for orotidine-5'-phosphate decarboxylase by in vivo functional screening. The results indicate that the conjugation system is useful in constructing G. kaustophilus libraries, which are practical in identifying thermophile genes.

Anthocyanin acyltransferases (AATs) catalyze a regiospecific acyl transfer from acyl-CoA to the glycosyl moiety of anthocyanins, thus playing an important role in flower coloration. The known AATs are subfamily members of an acyltransferase family, the BAHD family, which play important roles in secondary metabolism in plants. Here, we describe the purification, characterization, and cDNA cloning of a novel anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (hereafter referred to as Ss5MaT2) is a monomeric 46-kDa protein that catalyzes the transfer of the malonyl group from malonyl-CoA to the 4"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Thus, it is a malonyl-CoA:anthocyanin 5-glucoside 4"'-O-malonyltransferase. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA that encodes an acyltransferase protein. The steady-state transcript level of the gene was the highest in recently opened, fully pigmented flowers and was also correlated with the trend observed for an AAT gene responsible for the first malonylation step during salvianin biosynthesis. Immunoprecipitation studies using antibodies against the recombinant acyltransferase protein corroborated the identity of this cDNA as that encoding Ss5MaT2. The deduced amino acid sequence of Ss5MaT2 showed a low similarity (22-24% identity) to those of AATs and lacked the AAT-specific signature sequence. A phylogenetic analysis suggested that Ss5MaT2 is more related to acetyl-CoA:benzylalcohol acetyltransferase (BEAT) rather than to AAT. This is another example in which enzymes with similar, although not identical, substrate evolved from different branches of the BAHD family.

Complementary DNAs coding for two flavonol 3- O -glucoside malonyltransferases, Vh3MaT1 and Lp3MaT1 , were cloned from flowers of Verbena hybrida and Lamium purpureum , respectively, expressed in Escherichia coli cells, and functionally characterized. The cloning strategy took full advantage of the specific conservation of a sequence (–Tyr–Phe–Gly–Asn–Cys–, termed motif 2) in the anthocyanin-specific members of the versatile acyltransferase (VAT) family. Both of the expressed proteins, Vh3MaT1 and Lp3MaT1, effectively catalyzed the regiospecific transfer of the malonyl group from malonyl-CoA to the 6″-hydroxyl group of quercetin 3- O -glucoside and were highly specific for these acyl donor and acceptor. Therefore, these enzymes are malonyl-CoA:flavonol 3- O -glucoside-6″- O -malonyltransferases. Kinetic parameters were determined at pH 7.0 and 30 °C as follows: for Vh3MaT1, k cat , 2.9 s −1 ; k cat / K m for quercetin 3- O -glucoside, 17 s −1 mM −1 ; and k cat / K m for malonyl-CoA, 930 s −1 mM −1 ; for Lp3MaT1, k cat , 9.0 s −1 ; k cat / K m for quercetin 3- O -glucoside, 28 s −1 mM −1 ; and k cat / K m for malonyl-CoA, 360 s −1 mM −1 . These results suggested that Vh3MaT1 and Lp3MaT1 should serve as useful biocatalysts for the malonylation of quercetin 3- O -glucoside to control the bioactivities and pharmacokinetics of the flavonoid. The results also show that VAT members having motif 2 are not restricted to “anthocyanin” acyltransferases but should include an extended class of enzymes, i.e. “flavonoid glucoside” acyltransferases.

In the flowers of chrysanthemum (Dendranthema×morifolium), cyanidin 3-O-3″,6″-O-dimalonylglucoside is accumulated as a dominant anthocyanin. In this study, we isolated two full-length cDNAs from chrysanthemum flowers, Dm3MaT1 and Dm3MaT2, coding for enzymes that should be responsible for malonylation steps involved in the biosynthesis of the pigment. Both cDNAs were heterologously expressed in Escherichia coli cells, and the expressed products were functionally characterized. The Dm3MaT1 cDNA coded for a protein of 459 amino acid residues with a calculated molecular weight of 51,224, which showed >70% of sequence identity to anthocyanidin 3-O-glucoside-6″-O-malonyltransferases of Dahlia variabilis and Senecio cruentus. Specificity analyses of Dm3MaT1 further confirmed its identity as an anthocyanidin 3-O-glucoside-6″-O-malonyltransferase, which exclusively catalyzed the malonyl-CoA-dependent malonyl transfer to anthocyanidin 3-O-glucoside to produce anthocyanidin 3-O-6″-O-malonylglucoside. On the other hand, the Dm3MaT2 cDNA coded for a protein of 460 amino acid residues with a calculated molecular weight of 51,710, showing identities of 93% (for the nucleotide sequence) and 89% (for the deduced amino acid sequence) to Dm3MaT1. Unlike Dm3MaT1, the recombinant Dm3MaT2 catalyzed the consecutive dimalonyl transfer to anthocyanidin 3-O-glucoside; it produced anthocyanidin 3-O-6″-O-malonylglucoside from anthocyanidin 3-O-glucoside and could subsequently produce anthocyanidin 3-O-3″,6″-O-dimalonylglucoside from anthocyanidin 3-O-6″-O-malonylglucoside. Thus, Dm3MaT2 was the anthocyanidin 3-O-glucoside-3″,6″-O-dimalonyltransferase and should be responsible for the second malonylation of anthocyanins in the flower, providing the first example of anthocyanin acyltransferase that can catalyze the consecutive malony transfer.

Anthocyanins are responsible for the orange to blue coloration of flowers, fruits, and leaves. They are beneficial to human health and widely used as food colorants. Anthocyanin acyltransferases (AATs) are the plant enzymes that catalyze the regiospecific acyl transfer from acyl-CoA to the sugar moiety of anthocyanins. AATs are classified on the basis of their acyl-donor specificity into two categories; i.e. aliphatic and aromatic acyltransferases. However, the acyl-acceptor specificity of AAT differs greatly with the enzyme. Primary structural analyses of several AATs revealed that AATs form a subfamily within the versatile acyltransferase family and share highly conserved sequences such as motif 1 (-His-Xaa3-Asp-) and motif 3 (-Asp-Phe-Gly-Trp-Gly-) with each other. It is proposed that AAT-catalyzed acyl transfer proceeds with a general acid/base mechanism, where the enzyme and both acyl donor and acyl acceptor form a ternary complex before catalysis can occur. The histidine and aspartic acid residues located at motifs 1 and 3, respectively, appear to play very important roles during the proposed general acid/base catalysis. AAT cDNAs have been expressed in heterologous systems, providing a basis for applications of AATs in biotechnology, such as flower color modification and food colorant production by metabolic engineering of anthocyanin biosynthesis in plants.

“Cinerarin” is a polyacylated anthocyanin that is responsible for the blue coloration of cineraria (Senecio cruentus) flowers. We isolated a full-length cDNA (Sc3MaT) encoding a putative anthocyanin acyltransferase from S. cruentus. The Sc3MaT cDNA was expressed in Escherichia coli cells and the expression product was purified to homogeneity and functionally characterized. The Sc3MaT could catalyze the regiospecific malonyl transfer from malonyl-CoA (Km, 61μM) to pelargonidin 3-O-glucoside (Km, 11μM) to produce pelargonidin 3-O-6”-O-malonylglucoside with a kcat value of 8.8 s-1. The specificities for acyl donors and acceptors were highly restricted to malonyl-CoA and anthocyanidin 3-O-glucoside, respectively. Therefore, it may be concluded that Sc3MaT is a malonyl-CoA:anthocyanidin 3-O-glucoside-6”-O-malonyltransferase. The other enzymatic properties of Sc3MaT were comparable with those of known anthocyanin acyltransferases. Because a reaction product of Sc3MaT, delphinidin 3-O-6”-O-malonyglucoside, constitutes a part of cinerarin, Sc3MaT is probably involved in the cinerarin biosynthesis in this plant.

Microbes employ cell membranes for reducing exogenous stresses. Polyamino acid display on microbial cell surfaces and their effects on microbial chemical stress tolerance were examined. Growth analysis revealed that displays of polyarginine, polyaspartate and polytryptophan substantially enhanced tolerance of Escherichia coli to NaCl. A titration assay indicated that polyarginine and polyaspartate altered cell surface charges, implying tolerance enhancement via ion atmosphere and/or ionic bond network formations for electrostatic ion repulsion. The enhancement by polytryptophan may have arisen from surface hydrophobicity increase for hydrophobic ion exclusion, because of a strong correlation between hydrophobic characters of amino acids and their effects on tolerance enhancement. The display also enhanced tolerance to other salts and/or alcohols in E. coli and to NaCl in Saccharomyces cerevisiae. Thus polyamino acid display has the potential as an approach for conferring chemical stress tolerance on various microbes.

The orange to blue coloration of flowers in nature is, in most cases, provided by anthocyanins, a class of plant flavonoids, many of which are modified by malonyl group(s). However, the identity of the enzyme catalyzing the malonylation reaction remains to be established. Here, we describe for the first time the purification, characterization, and cDNA cloning of an anthocyanin malonyltransferase from scarlet sage (Salvia splendens) flowers. The purified enzyme (termed Ss5MaT1) was a monomeric 50-kDa protein catalyzing the regiospecific transfer of the malonyl group from malonyl-CoA to the 6"'-hydroxyl group of the 5-glucosyl moiety of anthocyanins. Ss5MaT1 showed a k(cat) value of 7.8 s(-1) at 30 degrees C and pH 7.0 for the malonylation of bisdemalonylsalvianin (pelargonidin 3-(6"-O-caffeyl-beta-glucopyranoside)-5-beta-glucopyranoside) and K(m) values of 101 microm and 57 microm for bisdemalonylsalvianin and malonyl-CoA, respectively. p-Coumaric acid, which mimics an aromatic acyl group linked to the 3-glucosidic moiety of an anthocyanin substrate, was a competitive inhibitor with respect to the substrate. This strongly suggests that the presence of an aromatic acyl group at the 3-glucosidic moiety of anthocyanin is important for substrate recognition by the enzyme. On the basis of the partial amino acid sequences of the purified enzyme, we isolated a cDNA encoding Ss5MaT1. Ss5MaT1 consisted of 462 amino acids and shared motifs that are commonly found among members of a versatile plant acyltransferase family, which was recently shown to include numerous homologs of unknown biochemical functions. Northern blot analysis revealed that the transcripts of Ss5MaT1 were detected in petals, sepals, bracts, and red stems, in accordance with the pigment accumulation patterns. Phylogenetic analysis suggests that the aliphatic and aromatic acylations of anthocyanins are generally catalyzed by subfamily members of the plant acyltransferase family.

On the basis of our study on the structure-activity relationships of 1,3,7-alkylxanthines and condensed-purines on cAMP-phosphodiesterase 4 (PDE 4) isoenzyme inhibitor, we investigated the synthesis and the inhibitory activity of 3-phenylxanthine and 4-phenyl[i]condensed-purine derivatives. Xanthines and condensed-purines with the phenyl group exhibited potent and selective PDE 4 inhibitory activity.

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5'-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura+ transformants with 1 microgram pRPPyr4 DNA and 1 x 10(7) viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0-1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.

A lithium ester enolate was activated by both a chiral etheral ligand and a lithium amide to form a ternary complex reagent that reacted with enoates giving the corresponding Michael addition products in reasonably high enantioselectivity of up to 97% ee.

Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.

Fumitremorgins (FTMs), tremorgenic mycotoxins produced by the human pathogen Aspergillus fumigatus, are prenylated indole alkaloids that have been extensively studied in view of their diverse chemical structures and biological activities. Their biosynthetic gene (ftm) cluster was identified on the basis of the genome sequence of A. fumigatus. However, it has been reported that the ftm cluster in genome reference strain Af293 is inactive, which makes complete understanding of the FTM pathway difficult. Hence, we used an FTM-producing strain of A. fumigatus, BM939, to dissect the FTM pathway. Here, we delineate the genetic determinant for the observed defect in the FTM pathway in A. fumigatus Af293. Metabolite profiling and sequence comparison of the two strains revealed a point mutation in ftmD as a possible cause of altered metabolite production in strain Af293. FTM production in Af293 was restored when a DNA fragment containing ftmD from BM939 was introduced. Biochemical analysis indicated that FtmD is a methyltransferase that catalyzes the conversion of 6-hydroxytryprostatin B into tryprostatin A. The mutated FtmD retained enzymatic activity but did not function under physiological conditions, resulting in blockage of the FTM pathway in A. fumigatus Af293.

The etiologies of idiopathic sudden sensorineural hearing loss (SSNHL) and Meniere's disease remain unclear. Recently, accumulating evidence has demonstrated that free radicals are related to the pathology of inner ear disease. Because genetic factors may contribute partly to the etiologies of SSNHL and Meniere's disease, we investigated the association between genetic polymorphisms located in genes related to the free-radical process and susceptibility to SSNHL and Meniere's disease. We compared 83 patients affected by SSNHL and 83 patients affected by Meniere's disease with 2048 adults (for SSNHL) and 1946 adults (for Meniere's disease) who participated in the National Institute for Longevity Sciences, Longitudinal Study of Aging. Multiple logistic regression was used to calculate odds ratios (ORs) for SSNHL and Meniere's disease in individuals with polymorphisms in the genes: methionine synthase (MTR; rs1805087); methionine-synthase reductase (MTRR; rs1801394); nitric oxide synthase 3 (NOS3; rs1799983); caveolin 1 (Cav1; rs3840634); melatonin receptor 1B (MTNR1B; rs1387153); NAD(P)H oxidase p22(phox) subunit (NADH/NADPHp22phox; rs4673); and mitochondria 5178 (MT5178; rs28357984). The NOS3 polymorphism was significantly associated with a risk of SSNHL; in addition, the OR for the NOS3 polymorphism and SSNHL risk was 2.108 (CI, 1.343-3.309) with adjustment for age and sex. The Cav1 polymorphism was significantly associated with a risk of Meniere's disease; moreover, the OR for the Cav1 polymorphism and Meniere's disease risk was 1.849 (CI, 1.033-3.310) with adjustment for age and sex. In conclusion, the NOS3 and Cav1 polymorphisms were significantly associated with the risk of SSNHL and Meniere's disease, respectively.

Laboratory experiments were performed in a climate simulator, where temperature, relative humidity, wind velocity and artificialinsolation can be controlled, to investigate man's physiologicalresponse and thermal comfort. The following results were obtained; 1) skin temperature decreases with wettedness, 2) variation ofmetabolic ratio with meteorological conditions can be ignorable and 3) thermal sensation can not be explained by increase of skintemperature, which conflicts with the former physiological models.

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (Δ pyrF ) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (Δ pyrF Δ pyrR ) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous β-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF -based counterselection system in a Bacillus -related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus . Our results also suggest a new strategy for establishment of counterselection systems.

Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, d-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His(6)-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.

DNA methylation in Streptomyces griseus IFO 13350 was analyzed by high-performance liquid chromatographic analysis and bisulfite-based analysis to reveal two methylation sites, 5'-GC5mCGGC-3' and 5'-GAG5mCTC-3'. The methylation was reconstituted in Escherichia coli by simultaneous expression of S. griseus SGR4675 and S. achromogenes M.SacI. The E. coli cells produced plasmids that mimicked the methylation profile of S. griseus DNA, which was readily introduced into S. griseus. The results of this study raise the possibility of a promising approach to establish efficient transformation in several streptomycetes.

The completion of more than ten fungal genomes has revealed the presence of an enormous number of genes that encode proteins typically involved in secondary-metabolite biosynthesis, such as polyketide synthase and nonribosomal peptide synthetase. These genes, which are responsible for forming the backbone structures of natural products, are often clustered with the genes encoding so-called tailoring enzymes, which are responsible for modifications of the metabolites, such as prenylation, methylation, and hydroxylation. Their gene organization allows us to predict biosynthetic products that arise from the corresponding gene clusters. Based on such predictions, many biosynthetic gene clusters and their metabolites have been identified by various approaches, such as heterologous expression, gene knockout and complementation analyses, and silent-gene activation. On the other hand, prediction of the function of each gene in the clusters is not always successful because only the types of protein functions can be annotated by a homology search; this is not sufficient for the correct prediction of substrates or products of unknown enzymes. Therefore, functional analyses of every gene in biosynthetic gene clusters are needed to discover the enzymes involved in important processes of natural product biosynthesis. The ftm cluster, which is responsible for fumitremorgin production in Aspergillus fumigatus (Figure S1A in the Supporting Information), is one of the secondary metabolite biosynthetic gene clusters whose products have been identified based on genomic information. Biochemical and genetic studies have demonstrated the functions of most of the ftm genes: an NRPS gene, ftmA ; two prenyltransferase genes, ftmB and ftmH (also known as ftmPT1 and ftmPT2) ; 6] and three cytochrome P450 genes, ftmC, ftmE, and ftmG. In spite of these extensive studies, there are still some missing steps in the biosynthetic pathway to fumitremorgins that are predicted to involve oxygenases. Our previous analysis of the ftm cluster revealed that three cytochrome P450s are responsible for key steps in the fumitremorgin biosynthetic pathway (Figure S1B), pointing out that the only remaining oxygenase gene, ftmF, which encodes a protein belonging to the a-ketoglutarate (a-KG)-dependent dioxygenases, is a candidate for such missing enzymes. Very recently, Steffan et al. showed that FtmF catalyzes the conversion of fumitremorgin B (1) to verruculogen (2) by using recombinant protein (Scheme 1). This biosynthetic step is unique in that FtmF catalyzes the formation of an endoperoxide bond between two isoprene moieties. However, no genetic evidence for the involvement of ftmF in the biosynthesis of 2 in A. fumigatus has been provided. The FtmF reaction itself remains to be fully understood due to the paucity of biochemical data. Here, we perform a knockout experiment and a kinetic analysis to clarify the function of FtmF as an endoperoxidation catalyst. Furthermore, metabolite profiling with a gene disruption mutant and the identification of FtmF reaction products reveal that FtmF plays an important role in giving rise to the structural diversity of fumitremorgins. We first compared the metabolite profiles of the ftmF disruption mutant to that of the wild-type strain. The DftmF strain was previously generated from the strain TAFK1.39 (DakuA ::ptrA) derived from a fumitremorgin-producer stain, BM939, by replacing the entire coding region with the hygromycin B-resistance gene cassette. The transformants were cultivated in order to analyze their metabolite profiles by LC/ ESI-MS. The disruption of ftmF stopped the production of 2 but had little or no effect on the production of 1 and its biosynthetic intermediates (Figure 1); this demonstrates that ftmF is involved in the biosynthesis of 2 in A. fumigatus. For biochemical characterization, the recombinant FtmF was expressed in Escherichia coli as a His6-tagged fusion protein and purified to homogeneity by metal ion affinity chromatography (Figure S2). Size-exclusion column chromatography sug

In grixazone biosynthesis by Streptomyces griseus, a key intermediate 3-amino-4-hydroxybenzoic acid (3,4-AHBA) is converted to another key intermediate 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL). Two genes griC and griD in the grixazone biosynthesis gene cluster were found to be responsible for this conversion, because disruption of each gene resulted in the extracellular accumulation of 3-acetylamino-4-hydroxybenzoic acid, a shunt product from 3,4-AHBA. Significant sequence similarity of GriC to AMP-binding proteins and of GriD to NAD(P)-dependent aldehyde dehydrogenases suggested that GriC and GriD constituted an ATP- and NAD(P)-dependent carboxylic acid reductase (CAR) catalyzing reduction of 3,4-AHBA to produce 3,4-AHBAL through acyl-AMP formation, as is found for the reactions catalyzed by some CARs. griG encoding a benzoate transporter homologue in the grixazone biosynthesis gene cluster was nonessential for grixazone biosynthesis but probably enhanced the membrane permeability for 3,4-AHBA. Simultaneous overexpression of griC, griD, and griG in S. griseus mutant cells deficient in an acetyltransferase responsible for N-acetylation of 3,4-AHBA led to efficient bioconversion of exogenously added 3,4-AHBA to 3,4-AHBAL. This system also turned out to be useful for reduction of some aryl carboxylates to the corresponding aryl aldehydes.

An arylamine N -acetyltransferase (NAT) responsible for the N acetylation of exogenous 3-amino-4-hydroxybenzoic acid in Streptomyces griseus was identified and characterized. This enzyme was distinct from other eukaryotic and bacterial NATs in that it acetylated various 2-aminophenol derivatives more effectively than it acetylated 5-aminosalicylic acid, and thus it may be involved in the metabolism of xenobiotic compounds.

The shikimate pathway, including seven enzymatic steps for production of chorismate via shikimate from phosphoenolpyruvate and erythrose-4-phosphate, is common in various organisms for the biosynthesis of not only aromatic amino acids but also most biogenic benzene derivatives. 3-Amino-4-hydroxybenzoic acid (3,4-AHBA) is a benzene derivative serving as a precursor for several secondary metabolites produced by Streptomyces, including grixazone produced by Streptomyces griseus. Our study on the biosynthesis pathway of grixazone led to identification of the biosynthesis pathway of 3,4-AHBA from two primary metabolites. Two genes, griI and griH, within the grixazone biosynthesis gene cluster were found to be responsible for the biosynthesis of 3,4-AHBA; the two genes conferred the in vivo production of 3,4-AHBA even on Escherichia coli. In vitro analysis showed that GriI catalyzed aldol condensation between two primary metabolites, l-aspartate-4-semialdehyde and dihydroxyacetone phosphate, to form a 7-carbon product, 2-amino-4,5-dihydroxy-6-one-heptanoic acid-7-phosphate, which was subsequently converted to 3,4-AHBA by GriH. The latter reaction required Mn(2+) ion but not any cofactors involved in reduction or oxidation. This pathway is independent of the shikimate pathway, representing a novel, simple enzyme system responsible for the synthesis of a benzene ring from the C(3) and C(4) primary metabolites.

Grixazone contains a phenoxazinone chromophore and is a secondary metabolite produced by Streptomyces griseus. In the grixazone biosynthesis gene cluster, griF (encoding a tyrosinase homolog) and griE (encoding a protein similar to copper chaperons for tyrosinases) are encoded. An expression study of GriE and GriF in Escherichia coli showed that GriE activated GriF by transferring copper ions to GriF, as has been observed for a Streptomyces melanogenesis system in which the MelC1 copper chaperon transfers copper ions to MelC2 tyrosinase. In contrast with tyrosinases, GriF showed no monophenolase activity, although it oxidized various o-aminophenols as preferable substrates rather than catechol-type substrates. Deletion of the griEF locus on the chromosome resulted in accumulation of 3-amino-4-hydroxybenzaldehyde (3,4-AHBAL) and its acetylated compound, 3-acetylamino-4-hydroxybenzaldehyde. GriF oxidized 3,4-AHBAL to yield an o-quinone imine derivative, which was then non-enzymatically coupled with another molecule of the o-quinone imine to form a phenoxazinone. The coexistence of N-acetylcysteine in the in vitro oxidation of 3,4-AH-BAL by GriF resulted in the formation of grixazone A, suggesting that the –SH group of N-acetylcysteine is conjugated to the o-quinone imine formed from 3,4-AHBAL and that the conjugate is presumably coupled with another molecule of the o-quinone imine. GriF is thus a novel o-aminophenol oxidase that is responsible for the formation of the phenoxazinone chromophore in the grixazone biosynthetic pathway.

To clarify the effect of 1-methyl-3-propyl-7-N,N-dimethylpropylamide-xanthine (MPDAX) on doxorubicin (DOX) transport, we examined the efficacy of MPDAX as an amplifier of the antitumor activity of DOX in mice bearing tumors with different properties as to DOX transport across cell membranes. MPDAX significantly enhanced the DOX-induced antitumor activity on DOX-sensitive tumors. It is expected that the increase in antitumor activity caused by MPDAX contributes to the increased DOX concentration in tumors due to the MPDAX-induced change in DOX transport via the transporter expressed in sensitive tumor cells. In contrast, in M5076, a lower sensitive to DOX, MPDAX decreased the tumor weight by half at an otherwise ineffective dose of DOX. Furthermore, in P388/DOX, DOX has no effect, but MPDAX caused an elevation of the DOX-induced antitumor activity with an increase in the DOX concentration in the tumors. The results suggested that MPDAX is a novel amplifier for antitumor agents as it significantly increased the antitumor activity toward tumors with different properties. The DOX concentrations in the MPDAX + DOX group for all tumor lines were about two-fold those in the DOX alone group. Furthermore, MPDAX and DOX exhibited significant inhibitory effects on uridine and thymidine uptake. It is known that nucleoside transporters increase the membrane permeability of DOX. We speculated that MPDAX inhibits the cell membrane transport of uridine and thymidine via nucleoside transporters. MPDAX, acting via nucleoside transporters, increases the DOX-induced antitumor activity toward many tumor types and is an useful biochemical modulator.

To reverse the adverse reactions of alkylxanthines and to develop novel inhibitors of cyclic AMP phosphodiesterase 4 (PDE4), a series of heterocycle [a]-, [b]-, [c,d]-, and [i]-condensed purines were designed and synthesized. Although all compounds did not display PDE1 and PDE3 inhibitory activities, several heterocycle [i]-condensed purines strongly inhibited PDE4. Especially, dl-3,4-dipropyl-8-methyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (dl-7c) exhibited comparable PDE4 inhibitory activity (IC(50)=1.9 microM) to rolipram and denbufylline (DBF).