Shigeru Mineki

Novel Mitochondrial Complex II Isolated from Trypanosoma cruzi Is Composed of 12 Peptides Including a Heterodimeric Ip Subunit

Mitochondrial respiratory enzymes play a central role in energy production in aerobic organisms. They differentiated from the α-proteobacteria-derived ancestors by adding noncatalytic subunits. An exception is Complex II (succinate: ubiquinone reductase), which is composed of four α-proteobacteria-derived catalytic subunits (SDH1-SDH4). Complex II often plays a pivotal role in adaptation of parasites in host organisms and would be a potential target for new drugs. We purified Complex II from the parasitic protist Trypanosoma cruzi and obtained the unexpected result that it consists of six hydrophilic (SDH1, SDH2N, SDH2C, and SDH5-SDH7) and six hydrophobic (SDH3, SDH4, and SDH8-SDH11) nucleus-encoded subunits. Orthologous genes for each subunit were identified in Trypanosoma brucei and Leishmania major. Notably, the iron-sulfur subunit was heterodimeric; SDH2N and SDH2C contain the plant-type ferredoxin domain in the N-terminal half and the bacterial ferredoxin domain in the C-terminal half, respectively. Catalytic subunits (SDH1, SDH2N plus SDH2C, SDH3, and SDH4) contain all key residues for binding of dicarboxylates and quinones, but the enzyme showed the lower affinity for both substrates and inhibitors than mammalian enzymes. In addition, the enzyme binds protoheme IX, but SDH3 lacks a ligand histidine. These unusual features are unique in the Trypanosomatida and make their Complex II a target for new chemotherapeutic agents.

Two forms of NAD-dependent D-mandelate dehydrogenase in Enterococcus faecalis IAM 10071.

ABSTRACT Two forms of NAD-dependent d-mandelate dehydrogenase (d-ManDHs) were purified from Enterococcus faecalis IAM 10071. While these two enzymes consistently exhibited high activity toward large 2-ketoacid substrates that were branched at the C3 or C4 position, they gave distinctly different Km and Vmax values for these substrates and had distinct molecular weights by gel electrophoresis and gel filtration.

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases produced by Agrobacterium sp. K-03

Abstract A poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Agrobacterium sp. strain K-03, isolated from soil, secreted two PHB depolymerases into the culture fluid when it was cultivated on PHB, 3-hydroxybutyrate (3HB), or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) comprising 43 mol% 3-hydroxyvalerate (P(3HB-co-43%3HV)) as the sole source of carbon. The two extracellular PHB depolymerases, designated E1 and E2, were purified from the culture fluid using DEAE-Toyopearl 650M column chromatography followed by gel filtration in Sephadex G-75 column chromatography. E1 and E2 are basic proteins having molecular weights of 46 and 44 kDa, and isoelectric points of 9.0 and 8.9, respectively. The optimal pHs of E1 and E2 for degradation of PHB are 8.1 and 7.9, respectively, and both of them have optimal PHB degradation temperatures of 45°C. Their enzymatic activities were considerably inhibited by dithiothreitol, phenylmethanesulfonyl fluoride, and Tween 20. The K m values of E1 and E2 for PHB were 17.8 μg/ml and 70.5 μg/ml, respectively. At pH 8.0 and 30°C, both enzymes completely degraded PHB, and they degraded 94% of P(3HB-co-43%3HV). The methyl ester of 3HB dimer was degraded by each enzyme to form the free dimer and the free monomer of 3HB.

Degradation of Polyaromatic Hydrocarbons by Fungi Isolated from Soil in Japan

There are fewer studies on the degradation of polyaromatic hydrocarbons (PAHs) by fungi than those by bacteria. Three fungi (strains A, B, and C), which could grow on pyrene as the sole source of carbon, were isolated from soils at the side of a freeway, a gas station, and a lake in Japan. Strain A was tentatively classified as members of the Trichoderma/Hypocrea genus, and both strains B and C were classified as belonging to the Fusarium genus on the basis of their base sequence of 18S ribosomal DNA and morphological characteristics. After an incubation period of 7 and 14 days at 28°C in an inorganic Czapek-Dox medium with PAHs as the substrate, both strains A and B assimilated anthracene and fluoranthene, whereas strain C assimilated neither of them. The pyrene-degrading activity and the growth of strain A improved by adding 0.02% yeast extract, 0.1% sucrose, or 0.1% lactose. The pyrene-degrading activity was also found to be more effective at pH 4 rather than at pH 6.5.

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity.

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.

Cloning, nucleotide sequencing, and expression in Escherichia coli of the gene for formate dehydrogenase of Paracoccus sp. 12-A, a formate-assimilating bacterium

The gene for the NAD-dependent formate dehydrogenase (FDH) of Paracoccus sp. 12-A, a formate-assimilating bacterium, was cloned through screening of the genomic library with activity staining. The FDH gene included an open reading frame of 1,200 base pairs, and encoded a protein of 43,757 Da, which had high amino acid sequence identity with known FDHs, in particular, with bacterial enzymes such as those of Moraxella sp. (86.5%) and Pseudomonas sp. 101 (83.5%). The gene was highly expressed in Escherichia coli cells using an expression plasmid with the pUC ori and tac promoter. The recombinant enzyme was somewhat inactive in the stage of the cell-free extract, but its activity markedly increased with purification, in particular, with the step of heat-treatment at 50°C. The purified enzyme showed essentially the same properties as the enzyme from the original Paracoccus cells.

Estimation of Indoor Air PAH Concentration Increases by Cigarette, Incense-Stick, and Mosquito-Repellent-Incense Smoke

Abstract Smoke from cigarette smoking and burning of incense sticks and mosquito-repellent incense, which is particularly used in summer, are thought to be primary factors of indoor air pollution by polycyclic aromatic hydrocarbons (PAHs) in Japanese houses. In this study, these respective smokes as indoor air pollutants were evaluated by quantifying their particulate matter (PM) and 7 PAHs. PM and PAHs in smoke from 5 popular brands of Japanese cigarettes were collected on the glass fiber filter by the international smoking mode. PM was quantified by weighing the filter before and after trapping them. PAHs were analyzed by HPLC after extracting the filter with ethanol/benzene (1:3, by vol.). Similarly, evaluation of smoke respectively generated by natural burning of 4 brands of incense sticks and 3 brands of mosquito-repellent incenses was also conducted. As the results of these experiments, the average concentrations of PM and benzo[a]pyrene in a 35 m3 closed room were estimated to increase 1630 μg/m3 and 8.2 ng/m3, respectively, by smoking of 3 cigarettes; 1320 μ/m3 and 4.1 ng/m3, respectively, by burning of 2 incense sticks; and 2510 μg/m3 and 17.3 ng/m3, respectively, by burning of 8 cm of mosquito-repellent incense.

A new cerebroside of the n-Alkane-assimilating yeast Candida deformans

Abstract A glucocerebroside was isolated from the n-alkane-assimilating yeast Candida deformans MIL 4041 and named “Deformin”. The cerebroside contained only a single sugar, glucose, attached to ceramide in a β-configuration, and only a single fatty acid, 2-hydroxypalmitic acid. The long-chain base was identified as (5E,9Z)-C19-sphinga-5,9-diene. Therefore, the structure of the cerebroside was determined to be (5E,9Z)-N-2′-hydroxypalmitoyl-1-O-β-glucopyranosyl-2-amino-5,9- nonadecadiene-1,3-diol. Deformin showed the fruiting-inducing activity of fungi.

Microbial hydroxylation of 11,13-dehydrosantonin by Aspergillus nigert☆

Abstract The microbial transformation of a sesquiterpene lactone, 11,13-dehydro-(−)-α-santonin (DS), was carried out by 7 fungi. Although 6 of the fungi transformed the substrate into three (one fungus) or fewer (5 fungi) kinds of products, Aspergillus niger MIL 5024 gave five products including all of those produced by the others. The products were identified by UV, IR, mass, and 1 H-NMR spectroanalyses as (−)-α-santonin (P1), 11-hydroxy-(−)-α-santonin (P2), 13-hydroxy-(−)-α-santonin (P3), 3,6,9-trihydroxy-9,10- seco -selina-1,3,5(10)-trien-12-oic acid- 12,6-lactone (P4), and 8- epi -artemisin (P5). P3, a new compound, was also produced by A. niger MIL 5035, but P4 and P5 were produced only by strain MIL 5024. The biosynthetic pathway of these 5 products from DS was estimated.

Microbial production of hydroxy-C19-steroids as estrogen synthetase (P-450 · aromatase) inhibitors

Abstract Four monohydroxy derivatives of androst-4-ene-3,17-dione (4AD) and two of androst-4-ene-3,6,17-trione (4AT), derived from microbial transformation of 4AD and 4AT, were purified and identified by UV, IR, mass, 13 C-NMR, and 1 H-NMR spectroanalyses. These derivatives were evaluated as inhibitors of human placental aromatase. They showed dose-dependent inactivation of aromatase in the presence of reduced nicotinamide adenine dinucleotide phosphate. Among them, 6α-hydroxyandrost-4-ene-3,17-dione (6α-OH-4AD) was the most potent, exhibiting 97.8% inhibition at 100 μM; this value was 1.04-fold higher than the inhibition shown by 4AT (93.6%). The other derivatives, 16β-OH-4AT, 15α-OH-4AD, 15β-OH-4AD, 6β-OH-4AD and 12α-OH-4AT, inhibited the aromatase 80.4%, 63.6%, 61.8%, 30.1%, and 9.6%, respectively, at the same concentration.