Osamu Johdo

The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome.

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 μM (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T. (1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug. An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 μm of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 position) was inhibited only 11 and 24%, respectively. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain. The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.

Insight into functional diversity of cytochrome P450 in the white-rot basidiomycete Phanerochaete chrysosporium: involvement of versatile monooxygenase.

To elucidate functional diversity of cytochrome P450 monooxygenases from the white-rot basidiomycete Phanerochaete chrysosporium (PcCYPs), we conducted a comprehensive functional screening using a wide variety of compounds. A functionomic survey resulted in characterization of novel PcCYP functions and discovery of versatile PcCYPs that exhibit broad substrate profiles. These results suggested that multifunctional properties of the versatile PcCYPs would play crucial roles in diversification of fungal metabolic systems involved in xenobiotic detoxification. To our knowledge, this is the first report describing multifunctional properties of versatile P450s from the fungal kingdom. An increased compilation of PcCYP functions will facilitate a thorough understanding of metabolic diversity in basidiomycetes and provide new insights that could also expedite practical applications in the biotechnology sector.

The Fibrinolytic Activity of a Novel Protease Derived from a Tempeh Producing Fungus, Fusarium sp. BLB

Tempeh is a traditional Indonesian soybean-fermented food produced by filamentous fungi, Rhizopus sp. and Fusarium sp. We isolated and sequenced the genomic gene and a cDNA clone encoding a novel protease (FP) from Fusarium sp. BLB. The genomic gene was 856 bp in length and contained two introns. An isolated cDNA clone encoded a protein of 250 amino acids. The predicted amino acid sequence of FP showed highest homology, of 76%, with that of trypsin from Fusarium oxysporum. The hydrolysis activity of FP toward synthetic peptide was higher than that of any other protease tested, including Nattokinases. Furthermore, the thrombolytic activity of FP was about 2.1-fold higher than that of Nattokinase when the concentration of plasminogen was 24 units/ml. These results suggest that FP is superior to Nattokinases in dissolving fibrin when absorbed into the blood.

Anthracycline metabolites from Streptomyces violaceus A262. III : new anthracycline obelmycins produced by a variant strain SE2-2385

New anthracycline antibiotics, designated as obelmycins A, D, E, F and G, were isolated from the culture broth of a variant strain of beta-rhodomycin-producing Streptomyces violaceus A262, identified as beta-isorhodomycinone glycosides and gamma-isorhodomycinone glycosides and assayed for their in vitro cytotoxicities against murine leukemic L1210 cell culture and the antimicrobial activities in comparison with some known anthracyclines.

Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS-PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.

Daunomycin biosynthesis by microbial conversion of precursor metabolites using biosynthetically blocked mutants

Abstract The biosynthetic pathway of daunomycin (DM) from intermediate aglycone aklavinone (AKN) is studied by biosynthetic conversion of various precursors using non-DM-producing blocked mutants. DM formation starts by (i) 11-oxidation of AKN to yield e-rhodomycinone (e-RMN) and is then achieved by at least six biosynthetic steps in the following order; (ii) 7-O-daunosaminylation of e-RMN to yield D788-6 (5A: 7-O-daunosaminyl e-RMN); (iii) 10-demethylesterification to yield D788-1 (RP: 10-carboxy-13-deoxocarminomycin); (iv) 10-decarboxylation to yield D788-11 (5B: 13-deoxocarminomycin); (v) 4-O-methylation to yield feudomycin A (13-deoxodaunomycin); (vi) 13-oxidation to yield 13-dihydrodaunomycin; (vii) 13-dehydrogenation to yield DM. However, the DM biosynthetic route via carminomycin is not observed. It is also found that most of these possible biosynthetic enzymes involved in DM production can catalyze some related metabolites other than their practical precursors.

Isolation and identification of a common intermediate produced by blocked mutants from β-rhodomycin and daunomycin producers

Biosynthetically blocked mutants isolated from the β-rhodomycin-producing Streptomyces violaceus A262 strain SC-7 and daunomycin-producing Streptomyces sp. D788 strain G1-1 were found to accumulate maggiemycin in their culture broths. Bioconversion tests using mutants that did not produce these antibiotics also proved that maggiemycin is a common precursor in the biosynthesis of β-rhodomycin and daunomycin.

New Anthracycline Antibiotic CG12 Obtained by Microbial Glycosidation of α-Citromycinone

Aiming at obtaining new antitumor anthracycline antibiotics, we have studied the production of hybrid anthracyclines by biosynthetic glycosidation of natural and semisynthetic anthracyclinones using antibiotic-negative mutants of anthracycline-producing microorganisms.1 ~3) We have prepared 16 hybrid anthracyclines,4 ~7) among which betaclamycins,7) trisarubicinol,5) and 2-hydroxyaclacino-