Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe²⁴⁶ (PsOYE)/Phe²⁵⁰ (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences.

Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.     

5-Halogenated thymidine analogues induce a senescence-like phenomenon in HeLa cells

We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.     

Single-molecule PCR using water-in-oil emulsion

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.     

Clustering of Human Chromosome Fragments on the Mouse Genome by Chromosome-Mediated Gene Transfer

Pieces of metaphase chromosomes prepared from mouse cells containingneo-tagged human chromosome 7 were transferred to mouse cellswith calcium phosphate to isolate G418-resistant clones. FISHanalysis revealed that the majority of them contained humanDNA at a single site on their genome. These transformants containedSTS markers mapped to various regions of chromosome 7. It isthus suggested that pieces of human chromosomes tend to assembleand integrate on the mouse genome.     

High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.     

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.     

Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ( [125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [125I]EGF in lysosomes and the disappearance of [125I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not beta-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 micrograms/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.     

Stereospecific Reduction of Ethyl 2′ -Ketopantothenate to Ethyl D-(+)-Pantothenate with Microbial Cells as a Catalyst

A novel enzymatic process for the synthesis of D-(+)-pantothenic acid through the asymmetric reduction of the 2′ -ketopantothenate ester is described. Candida macedoniensis AKU 4588 was found to convert ethyl 2′ -ketopantothenate (80 mg/ml) almost specifically to ethyl D-(+)-pantothenate (>98% enantiomeric excess), with a molar yield of 97.2%.