Cloning of the pyridoxine 5'-phosphate phosphatase gene (pdxP) and vitamin B6 production in pdxP recombinant Sinorhizobium meliloti.

A novel gene (pdxP) encoding a pyridoxine 5'-phosphate (PNP) phosphatase involved in the last step of pyridoxine biosynthesis was cloned from Sinorhizobium meliloti IFO 14782 on the basis of the peptide sequences of the natural enzyme. The pdxP gene is an open reading frame (708 bp) encoding 235 amino acid residues with a calculated molecular weight of 26,466. From its deduced amino acid sequence, it was predicted that the enzyme belongs to the haloacid dehalogenase superfamily.

Flavin adenine dinucleotide-dependent 4-phospho-D-erythronate dehydrogenase is responsible for the 4-phosphohydroxy-L-threonine pathway in vitamin B6 biosynthesis in Sinorhizobium meliloti.

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase.

Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid.

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium.

Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.

A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found.

Purification and characterization of pyridoxine 5'-phosphate phosphatase from Sinorhizobium meliloti.

Here we report the purification and biochemical characterization of a pyridoxine 5'-phosphate phosphatase involved in the biosynthesis of pyridoxine in Sinorhizobium meliloti. The phosphatase was localized in the cytoplasm and purified to electrophoretic homogeneity by a combination of EDTA/lysozyme treatment and five chromatography steps. Gel-filtration chromatography with Sephacryl S-200 and SDS/PAGE demonstrated that the protein was a monomer with a molecular size of approximately 29 kDa.

Piperazine propanol derivative as a novel antifungal targeting 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan synthase, which synthesizes a main component of fungal cell wall, is one of the promising targets for antifungal agents. In order to identify novel chemical classes of 1,3-beta-D-glucan synthase inhibitors, we screened a chemical library monitoring inhibition of the Candida albicans 1,3-beta-D-glucan synthase activity. The piperazine propanol derivative GSI578 [(2,6-difluoro-phenyl)-carbamic acid 3-(4-benzothiazol-2-yl-piperazine-1-yl)-propyl ester] was identified as a potent inhibitor against 1,3-beta-D-glucan synthase with an IC50 value of 0.

Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase.

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo.

Simultaneous determination of bioactive conformations and alignment rules by multi-way PLS modeling.

In this study, we propose a new three-dimensional quantitative structure-activity relationship (3D-QSAR) method for selecting bioactive conformations and alignment rule simultaneously. The possible conformations of all molecules are generated by conformational analysis and they are superimposed on template conformer with possible alignment rules. The field variables are calculated as 3D descriptor of structures.

Augmentation of the antitumor activity of capecitabine by a tumor selective dihydropyrimidine dehydrogenase inhibitor, RO0094889.

Capecitabine is an orally available fluoropyrimidine and is finally converted to 5-FU selectively in tumor tissues. In our study, we examined whether the antitumor activity of capecitabine is directly affected by a modulation of dihydropyrimidine dehydrogenase (DPD). The modulations were carried out by the overexpression of DPD in tumor cells and by tumor selective DPD inhibition.

Membrane-bound D-sorbitol dehydrogenase of Gluconobacter suboxydans IFO 3255--enzymatic and genetic characterization.

Gluconobacter strains effectively produce L-sorbose from D-sorbitol because of strong activity of the D-sorbitol dehydrogenase (SLDH). L-sorbose is one of the important intermediates in the industrial vitamin C production process. Two kinds of membrane-bound SLDHs, which consist of three subunits, were reportedly found in Gluconobacter strains [Agric.

The crystal structure and stereospecificity of levodione reductase from Corynebacterium aquaticum M-13.

The (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase (LVR) of the soil isolate bacterium Corynebacterium aquaticum M-13 is a NAD(H)-linked enzyme that catalyzes reversible oxidoreduction between (4R)-hydroxy-(6R)-2,2,6-trimethylcyclohexanone (actinol) and levodione. Here the crystal structure of a ternary complex of LVR with NADH and its inhibitor 2-methyl-2,4-pentanediol has been determined by molecular replacement and refined at 1.6-A resolution with a crystallographic R factor of 0.

Design and synthesis of the tumor-activated prodrug of dihydropyrimidine dehydrogenase (DPD) inhibitor, RO0094889 for combination therapy with capecitabine.

A series of tumor-activated prodrugs of the inhibitors of dihydropyrimidine dehydrogenase (DPD), an enzyme catabolizing 5-fluorouracil (5-FU: 4g), has been designed and synthesized. RO0094889 (11c) is a prodrug of 5-vinyluracil (4c), a known DPD inhibitor, and was designed to generate 4c selectively in tumor tissues by sequential conversion of 11c by three enzymes: esterase, cytidine deaminase and thymidine phosphorylase, the latter two of which are known to be highly expressed in various tumor tissues. When capecitabine (1), a tumor-activated prodrug of 5-FU, was co-administered orally with 11c, 5-FU in tumor tissues was significantly increased with only a slight increase of 5-FU in plasma as compared with oral capecitabine alone.

[Optimal dosing schedule in combination therapy with irinotecan and doxifluridine in a human colorectal cancer xenograft model].

A combination therapy with CPT-11 and 5-FU/LV has been recently established as a first-line therapy for metastatic colorectal cancer. However, severe adverse effects have also been reported from this combination therapy, and a modality to reduce the adverse effects is desired. 5'-DFUR, a pro-drug of 5-FU, shows less myelotoxicity than 5-FU, and thus it may be a better partner to combine with CPT-11.

The localization change of Ybr078w/Ecm33, a yeast GPI-associated protein, from the plasma membrane to the cell wall, affecting the cellular function.

The YBR078W/ECM33 gene of Saccharomyces cerevisiae encodes a glycosylphosphatidylinositol (GPI)-attached protein and its disruptant strain exhibited a temperature-sensitive (ts) growth defect. A HA-tagged Ybr078w protein, which complemented the ts growth phenotype of the ybr078wdelta strain, was predominantly located on the plasma membrane by GPI anchoring. To examine the requirement of the GPI anchoring on the plasma membrane for the function, the omega-minus region of Ybr078w was replaced with those of Ydr534c/Fit1 and Ynl327w/Egt2, which are known as GPI-dependent cell wall proteins.

Main polyol dehydrogenase of Gluconobacter suboxydans IFO 3255, membrane-bound D-sorbitol dehydrogenase, that needs product of upstream gene, sldB, for activity.

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.

Design, synthesis and antifungal activity of a novel water soluble prodrug of antifungal triazole.

A highly potent water soluble triazole antifungal prodrug, RO0098557 (1), has been identified from its parent, the novel antifungal agent RO0094815 (2). The prodrug includes a triazolium salt linked to an aminocarboxyl moiety, which undergoes enzymatic activation followed by spontaneous chemical degradation to release 2. Prodrug 1 showed high chemical stability and water solubility and exhibited strong antifungal activity against systemic candidiasis and aspergillosis as well as pulmonary aspergillosis in rats.

Biotin synthase of Bacillus subtilis shows less reactivity than that of Escherichia coli in in vitro reaction systems.

The biotin synthases of Bacillus subtilis and Escherichia coli were compared in a physiological reduction system using cell-free extracts and in a artificial reduction system using photo-reduced deazariboflavin. The biotin synthase of B. subtilis was less active than that of E.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 3.

A new series of acid-stable antifungal agents having strong inhibitory activity against Candida albicans N-myristoyltransferase (CaNmt) has been developed starting from acid-unstable benzofuranylmethyl aryl ether 2. The inhibitor design is based on X-ray crystallographic analysis of a CaNmt complex with aryl ether 3. Among the new inhibitors, pyridine derivative 8b and benzimidazole derivative 8k showed clear antifungal activity in a murine systemic candidiasis model.

Crystal structures of Candida albicans N-myristoyltransferase with two distinct inhibitors.

Myristoyl-CoA:protein N-myristoyltransferase (Nmt) is a monomeric enzyme that catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine residue of a variety of eukaryotic and viral proteins. Genetic and biochemical studies have established that Nmt is an attractive target for antifungal drugs. We present here crystal structures of C.

Excitatory amino acid-elicited tonic convulsions in mice and N-methyl-D-aspartate receptor activation: role of Ca(2+) influx and involvement of intracellular Ca(2+)-dependent biochemical processes.

Intravenously administered nimodipine (an L-type Ca(2+) antagonist) as well as dizocilpine (an N-methyl-D-aspartate--NMDA--antagonist) showed a wide spectrum of anticonvulsant activity in intracerebroventricular mouse models for excessive activation of excitatory amino acid receptors. The duration of Bay k-8644 (L-type Ca(2+) agonist; intracerebroventricular administration) caused seizures was significantly reduced by intravenously administered nimodipine. Intracisternal administration of Bay k-8644 lowered the convulsion threshold of an intracerebroventricular injection of NMDA.

Synthesis of novel water soluble benzylazolium prodrugs of lipophilic azole antifungals.

Water soluble N-benzyltriazolium or N-benzylimidazolium salt type prodrugs of several highly lipophilic triazole or imidazole antifungals have been synthesized. They were designed to undergo an enzymatic activation followed by a self-cleavage to release a parent drug. The prodrugs such as 16 had enough chemical stability and water solubility for parenteral use and were rapidly and quantitatively converted to the active substance in human plasma.

Differential sensitivity between Fks1p and Fks2p against a novel beta -1,3-glucan synthase inhibitor, aerothricin3 [corrected].

Fks1p and Fks2p are catalytic subunits of beta-1,3-glucan synthase, which synthesize beta-1,3-glucan, a main component of the cell wall in Saccharomyces cerevisiae. Although Fks1p and Fks2p are highly homologous, sharing 88.1% identity, it has been shown that Fks2p is more sensitive than Fks1p to one of echinocandin derivatives, which inhibits beta-1,3-glucan synthase activity.

Mapping of early firing origins on a replication profile of budding yeast.

Background: Understanding of the firing time determination of replication origins in the entire genome will require a genome-wide survey of replication origins and their mapping on chromosomes. A microarray technology was applied to obtain a genome-wide profile of DNA replication and to classify early firing origins.

Results: A total of 260 potential replication origins (PROs) were identified in the entire budding yeast genome: 247 as defined peaks on the replication profile and 13 as regions located in the chromosomal termini.

Cassette dosing approach and quantitative structure-pharmacokinetic relationship study of antifungal N-myristoyltransferase inhibitors.

Pharmacokinetic (PK) parameters of N-myristoyltransferase (Nmt) inhibitors were measured, and a multivariate quantitative structure-pharmacokinetic relationship (QSPKR) model for predicting rat elimination half-life (t(1/2)) values was constructed. One hundred seven benzofuran derivatives have been selected as the data set for QSPKR analysis. The correlation between the t(1/2) values and 30 physicochemical descriptors was examined by a stepwise multiple linear regression method.

5-Fluorouracil-induced intestinal toxicity: what determines the severity of damage to murine intestinal crypt epithelia?

To elucidate the relationship between 5-fluorouracil (5-FU) distribution and 5-FU-induced apoptosis and/or cell cycle arrest, microautoradiography was applied to murine intestinal crypts exposed to [14C] 5-FU by intravenous infusion. The histologic location of apoptotic cells in the crypt did not correlate to that of 5-FU. The temporal profiles of apoptotic and/or mitotic indexes corresponded to those of orally administered 5-FU in a previous study.