Shingo Fujisaki

Polyprenyl diphosphate synthase essentially defines the length of the side chain of ubiquinone

Ubiquinone, known as a component of the electron transfer system in many organisms, has a different length of the isoprenoid side chain depending on the species, e.g., Escherichia coli, Saccharomyces cerevisiae and humans have 8, 6, and 10 isoprene units in the side chain, respectively. No direct evidence has yet shown what factors define the length of the side chain of ubiquinone. Here we proved that the polyprenyl diphosphate that was available in cells determined the length of the side chain of ubiquinone. E. coli octaprenyl diphosphate synthase (IspB) was expressed with the mitochondrial import signal in S. cerevisiae. Such cells produced ubiquinone-8 in addition to the originally existing ubiquinone-6. When IspB was expressed in a S. cerevisiae COQ1 defective strain. IspB complemented the defect of the growth on the non-fermentable carbon source. Those cells had the activity of octaprenyl diphosphate synthase and produced only ubiquinone-8. These results opened the possibility of producing the type of ubiquinone that we need in S. cerevisiae simply by expressing the corresponding polyprenyl diphosphate synthase.

Molecular cloning and heterologous expression of the C-13 phenylpropanoid side chain-CoA acyltransferase that functions in Taxol biosynthesis

The structural pharmacophore of Taxol, responsible for binding the N terminus of the β-subunit of tubulin to arrest cell proliferation, comprises, in part, the 13-O-(N-benzoyl-3-phenylisoserinoyl) side chain. To identify the side chain transferase of Taxol biosynthesis, a set of transacylases obtained from an enriched cDNA library (constructed from mRNA isolated from Taxus cuspidata cells induced with methyl jasmonate for Taxol production) was screened. A cDNA clone (designated TAX7) encoding a taxoid C-13 O-phenylpropanoyltransferase was isolated which yielded a recombinant enzyme that catalyzes the selective 13-O-acylation of baccatin III with β-phenylalanoyl CoA as the acyl donor to form N-debenzoyl-2′-deoxytaxol. This enzymatic product was converted to 2′-deoxytaxol by chemical N-benzoylation, and the identity of this derivative was confirmed by spectrometric analyses. The full-length cDNA has an ORF of 1,335 bases and encodes a 445-aa protein with a calculated molecular weight of 50,546. Evaluation of kinetic parameters revealed Km values of 2.4 ± 0.5 μM and 4.9 ± 0.3 μM for baccatin III and β-phenylalanoyl-CoA, respectively. The pH optimum for the recombinant O-(3-amino-3-phenylpropanoyl)transferase is at 6.8. Identification of this clone completes acquisition of the five aroyl/acyltransferases involved in the biosynthesis of Taxol. Application of these transacylase genes in suitable host cells can improve the production yields of Taxol and could enable the preparation of second-generation Taxol analogs possessing greater bioactivity and improved water solubility.

Yeast Saccharomyces cerevisiae has two cis‐prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis

Background Dolichol is a family of long‐chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis‐prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established.

Involvement of an Essential Gene, mviN, in Murein Synthesis in Escherichia coli

We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Overexpression of the Saccharomyces cerevisiae MET17/MET25 gene in Escherichia coli and comparative characterization of the product with O-acetylserine O-acetylhomoserine sulfhydrylase of the yeast

The Saccharomyces cerevisiae MET17/MET25 gene encoding O-acetyl-L-serine (OAS)·O-acetyl-L-homoserine (OAH) sulfhydrylase (EC 4.2.99.10) was overexpressed in Escherichia coli and the gene product was purified to homogeneity, using three steps, with a recovery of 28% from the total cell extract. The gene product has been compared with OAS·OAH sulfhydrylase purified from the yeast cells. These two protein preparations were indistinguishable with respect to their behavior in polyacrylamide gel electrophoresis, both with and without sodium dodecyl sulfate, their specificity for substrate amino acids, Michaelis constant (Km) value for OAH, sensitivity to carbonyl reagents, absorption spectrum, isoelectric point, behavior in HPLC (both ion-exchange chromatography and gel filtration), sensitivity to heat treatment, susceptibility to trypsin digestion, and their N-terminal amino acid sequence. The results obtained imply that the gene product is properly processed in E. coli, and the technique developed in this study to overexpress the gene in bacterial cells provides us with a large amount of the purified preparation of the enzyme. In contrast to a previous report we found that cystathionine γ-lyase of S. cerevisiae behaved differently from OAS·OAH sulfhydrylase during the purification procedure.

Identification of tomoregulin-1 as a novel addicsin-associated factor.

Addicsin is a novel factor encoding a 23-kDa hydrophobic protein that is highly upregulated in the amygdala nuclei of morphine-administered mice. It is a murine homolog of human JWA and rat glutamate transporter-associated protein 3-18 (GTRAP3-18), a negative modulator of the neural glutamate transporter excitatory amino acid carrier 1 (EAAC1). Recent findings demonstrated that addicsin participates in various physiological processes by forming hetero- or homomultimeric complexes. However, the binding targets and molecular functions of addicsin remain largely unknown. To identify potential factors that associate with mouse addicsin, we performed a yeast two-hybrid screen using a 17-day-old mouse whole embryo cDNA library. We identified tomoregulin-1 (TR1) as a novel addicsin-associated factor. TR1, a type I transmembrane protein containing two follistatin-like modules and an epidermal growth factor-like domain, participates in nodal and bone morphogenetic protein signaling. Immunoprecipitation assays demonstrated that TR1 bound to addicsin, and that amino acids 145-188 of addicsin and amino acids 228-266 of TR1 were important for the formation of the addicsin-TR1 heterocomplex. The double-fluorescent immunohistochemical analysis revealed that addicsin and TR1 were coexpressed in neurons in the mature mouse brain regions tested. Moreover, TR1 showed a punctuate pattern throughout the cell, with preferential expression on the cell surface when expressed alone. However, TR1 predominantly redistributed to the endoplasmic reticulum (ER) when coexpressed with addicsin. Furthermore, coexpression of an addicsin mutant that lacked TR1 binding ability had little effect on the distribution of TR1. Biotinylation assays showed that coexpression of addicsin with TR1 suppressed the cell surface expression of TR1. Wound-healing assays demonstrated that the interaction of addicsin with TR1 had a significant effect on cell migration. These findings demonstrate that addicsin in the ER controls intracellular TR1 trafficking from the ER to plasma membrane and regulates cell migration through its interaction with TR1.

Molecular Characterization of Fluoroquinolone-Resistant Moraxella catarrhalis Variants Generated In Vitro by Stepwise Selection

ABSTRACT Moraxella catarrhalis causes respiratory infections. In this study, fluoroquinolone-resistant strains were selected in vitro to evaluate the mechanism of fluoroquinolone resistance. Strains with reduced fluoroquinolone susceptibility were obtained by stepwise selection in levofloxacin, and fluoroquinolone targets gyr and par were sequenced. Six novel mutations in GyrA (D84Y, T594dup, and A722dup), GyrB (E479K and D439N), and ParE (Q395R) involved in M. catarrhalis resistance to fluoroquinolones were revealed.

Non-invasive measurement of melanin-derived radicals in living mouse tail using X-band EPR

The aim of this experiment is to measure in vivo generation of melanin-derived radicals non-invasively, as a quantifiable index of radio-biological effect. Melanin-derived radicals in a living intact mouse tail tip were non-invasively measured in very simple way using an X-band electron paramagnetic resonance spectrometer. Colored mouse strains, C57BL/6NCr, BDF1, and C3H/He, have clear EPR signal corresponding to melanin-derived radicals in the tail tip; however, albino mouse strains, BALB/cCr, ddY, ICR, have no EPR signals. An X-ray fraction of 2 Gy/day (1 Gy/min) was repeatedly irradiated to a C3H/He mouse tail skin every Monday to Friday for 4 weeks. In comparison to before starting irradiation, the C3H/He mouse tail skin became darker, like a suntan. The melanin-derived radicals in C3H/He mouse tail skin were increased in association with X-ray fractions. Melanin-derived radicals in mouse tail skin can be readily and chronologically measurable by using X-band EPR spectrometer, and can be a marker for a radiobiological effect in the skin.

Analysis of six new genes encoding lysis proteins and coat proteins in Escherichia coli group A RNA phages.

Group A RNA phages consist of four genes—maturation protein, coat protein, lysis protein and replicase genes. We analyzed six plasmids containing lysis protein genes and coat protein genes of Escherichia coli group A RNA phages and compared their amino acid sequences with the known proteins of E. coli (group A), Pseudomonas aeruginosa (PP7) RNA phages and Rg‐lysis protein from Qβ phage. The size of lysis proteins was different by the groups but the coat proteins were almost the same size among phages. The phylogenetic analysis shows that the sub‐groups A‐I and A‐II of E. coli RNA phages were clearly dispersed into two clusters.