Cloning and sequencing of the aculeacin A acylase-encoding gene from Actinoplanes utahensis and expression in Streptomyces lividans.

Aculeacin A acylase (AAC), produced by Actinoplanes utahensis, catalyzes the hydrolysis of the palmitoyl moiety of the antifungal antibiotic, aculeacin A. Using mixed oligodeoxyribonucleotide probes based on the N-terminal amino acid (aa) sequences of the two subunits of AAC, overlapping clones were identified in a cosmid library of A. utahensis DNA. After the sub-cloning of a 3.0-kb fragment into Streptomyces lividans, the recombinant produced AAC extracellularly. The nucleotide sequence of this fragment predicted an open reading frame of 2358 bp with GTG start and TGA stop codons. The deduced 786-aa sequence should correspond to a single polypeptide chain, indicating that this polypeptide is processed to the active form which is composed of the two subunits. Threefold more AAC was obtained from the S. lividans recombinant carrying the cloned gene than the original A. utahensis strain.

Cloning of the gene encoding avermectin B 5-O-methyltransferase in avermectin-producing Streptomyces avermitilis.

Complementation of a mutant lacking avermectin B 5-O-methyltransferase (AveD) of Streptomyces avermitilis, which catalyses the methylation of the hydroxyl group at the C5 position of avermectin B compounds, revealed that the gene encoding AveD is in a 1.25-kb SalI-EcoNI fragment in the left region of the gene cluster for avermectin biosynthesis. The nucleotide sequence of this fragment predicted a 283-aa gene product homologous to several methyltransferases requiring S-adenosyl-l-methionine as a cofactor. After cloning of the aveD region from mutant not producing AveD, the complementation experiments were performed using a pair of hybrid fragments (AveD+/AveD- and AveD-/AveD+). They suggest that the mutation(s) is in the N-terminus of AveD. SSCP analysis of amplified DNA of the aveD region derived from both wild type and mutant strains supports the results of the complementation experiments. Sequence analysis of the aveD region of the mutant strain revealed that a point mutation is within ORF, being Thr23-->Ile substitution. This mutation causes the inactivation of O-methyltransferase activity of AveD.

A deacylation enzyme for aculeacin A, a neutral lipopeptide antibiotic, from Actinoplanes utahensis: purification and characterization.

An enzyme, tentatively termed aculeacin A acylase, useful in preparing deacylated peptides which are used as starting material for semisynthetic antifungal antibiotics, was purified from the culture filtrate of Actinoplanes utahensis NRRL12052. The purification involved ultrafiltration and column chromatographies on DEAE-cellulose, hydroxyapatite, and Butyl-Toyopearl 650M. The purified enzyme was composed of two dissimilar subunits with molecular weights of 55,000 and 19,000. The subunits were dissociated in the presence of 0.1% SDS or 6 M guanidine hydrochloride; the dissociation accompanied loss of acylase activity. The enzyme was fully active at pH 7.0 and at 60 degrees C. Its pI was estimated to be above 10.25. The Km and Vmax for aculeacin A were 1.53 mM and 39.7 mumol/min/mg-protein, respectively.

Ribosome-binding activities and antimicrobial activities of tylosin and its related compounds.

Structure-activity relationships of tylosin and related compounds were evaluated in terms of their antimicrobial and ribosome-binding activities. Demycarosyl derivatives, demycarosyltylosin and 20-deoxydemycarosylrelomycin, were slightly weaker than tylosin and 20-deoxyrelomycin, respectively, both in antimicrobial activity and in affinity to ribosomes. The corresponding demycarosyl-demycinosyl derivatives had weaker antimicrobial activities despite their relatively high affinities to ribosomes. A 23-deoxy-demycarosyl-demycinosyl derivative, 20-oxo-5-O-mycaminosylprotylonolide, had a higher affinity to ribosomes than that of tylosin and was equivalent to tylosin in antimicrobial activity against Gram-positive bacteria. These results suggest that the mycinose moiety increases the ability of the molecule to enter bacterial cells. Among the derivatives tested, a 23-iodo derivative, 20-deoxy-23-iodo-5-O-mycarosyltylonolide, had the highest affinity for ribosomes as well as the highest antimicrobial activity.

Chemical modification of tylosin: synthesis of amino derivatives at C-20 position of tylosin and demycarosyltylosin.

Reductive aminations of the aldehyde group at C-20 position of tylosin and demycarosyltylosin (desmycosin) were carried out using primary and secondary amines in the presence of sodium cyanoborohydride. Some of these derivatives brought about higher antimicrobial and ribosome-binding activities, and the structure-activity relationship is discussed.

Binding of [3H]tetrahydroleucomycin A3 to Escherichia coli ribosomes and the effect of 3"-O-acyl derivatives of leucomycins on the binding.

The analysis of [3H]tetrahydroleucomycin A3 binding to Escherichia coli ribosomes are described. The dissociation constant for tetrahydroleucomycin A3 binding to ribosomes was 1.15 x 10(-8) M. One molecule of tetrahydroleucomycin A3 was bound to each 70 S ribosome (50 S subunit) as reported with erythromycin. The effect of leucomycins and their 3"-O-acyl derivatives on [3H]tetrahydroleucomycin A3 binding to ribosomes was examined. In general, 3"-O-acyl derivatives of leucomycins exhibited stronger antimicrobial activity against Gram-positive bacteria and weaker (or equivalent) activity against E. coli than their mother compounds. However, the affinities to ribosomes were approximately equivalent to those of the mother compounds, suggesting that Gram-positive bacterial cells are more permeable to 3"-O-acyl derivatives than to the mother compounds.

Studies on bacterial cell wall inhibitors. X. Properties of phosph-N-acetylmuramoyl-pentapeptide-transferase in peptidoglycan synthesis of Bacillus megaterium and its inhibition by amphomycin.

The phospho-N-acetylmuramoyl-pentapeptide-transferase from Bacillus megaterium KM was characterized by the transfer reaction. The particulate enzyme preparation had the activity to transfer phospho-N-acetylmuramoyl-pentapeptide from UDP-N-acetylmuramoyl-pentapeptide to undecaprenoid-1-ol-phosphate. The optimum pH for activity was about 8.5. The reaction required the presence of Mg2+ and an SH-protector. With 25 mm Mg2+ the maximum activity was observed. The reaction was reversible and so the addition of UMP decreased the formation of undecaprenoid-1-ol-diphospho-N-acetylmuramoyl-pentapeptide. Amphomycin inhibited non-competitively the transferase for the substrate UDP-N-acetylmuramoyl-pentapeptide.

Pepticinnamins, new farnesyl-protein transferase inhibitors produced by an actinomycete. I. Producing strain, fermentation, isolation and biological activity.

Pepticinnamins A, B, C, D, E and F, a family of farnesyl-protein transferase (FPT) inhibitors were isolated from the fermentation broth of Streptomyces sp. OH-4652. These inhibitors were purified from whole broth by extraction with chloroform, followed by silica gel column chromatography, Sephadex LH-20 chromatography and reverse phase HPLC. Among these, pepticinnamin C showed the most potent inhibition (IC50-100 nM).

Pepticinnamins, new farnesyl-protein transferase inhibitors produced by an actinomycete. II. Structural elucidation of pepticinnamin E.

Structure of novel farnesyl transferase inhibitor, pepticinnamin E, is elucidated by NMR study. Pepticinnamin E is composed of five amino acids and o-pentenylcinnamic acid, having a molecular weight of 907. C-terminal glycylserine of the compounds is in the cyclized diketopiperazine form.

Andrastins A-C, new protein farnesyltransferase inhibitors produced by Penicillium sp. FO-3929. II. Structure elucidation and biosynthesis.

The structures of new protein farnesyltransferase inhibitors, andrastins A-C, were elucidated. The cyclopentane ring of andrastins exhibited keto-enol tautomerism, which made the structure hard to elucidate. Therefore, the structure of andrastin A was elucidated by INADEQUATE and 13C-13C couplings using 13C-labeled andrastin A. The absolute configuration of the p-bromobenzoyl derivative of andrastin A was elucidated by X-ray crystallographic analysis and its skeleton was shown to be ent-5 alpha,14 beta-androstane. The biosynthesis of andrastin A was also studied by the incorporation of 13C-labeled acetates. Though the andrastins had a common androstane skeleton, they were biosynthesized from a sesquiterpene and a tetraketide.

New brominated and halogen-less derivatives and structure-activity relationship of azaphilones inhibiting gp120-CD4 binding.

Novel brominated and halogen-less azaphilone (oxoisochromane) derivatives, 5-bromoochrephilone and dechloroisochromophilone IV, and known derivatives, dechloroisochromophilone III and isorotiorin, were isolated from the culture broth of a producing organism of isochromophilones I and II (azaphilones inhibiting gp120-CD4 binding), Penicillium multicolor FO-2338, fermented in a medium containing potassium bromide. Nineteen azaphilone-related compounds isolated from the above strain and from other fungi were tested for the inhibition of gp120-CD4 binding and the structure-activity relationship is discussed. Consequently, 5-bromoochrephilone is the strongest inhibitor (IC50, 2.5 microM). A halogen atom at C-5, a proton at C-8 and a diene structure in C-3 side chain of 6-oxoisochromane ring are necessary for gp120-CD4 binding.

Funalenone, a novel collagenase inhibitor produced by Aspergillus niger.

Funalenone, a phenalene compound that inhibits type I collagenase (MMP-1), was isolated from mycelium of Aspergillus niger FO-5904 by solvent extaction, ODS column chromatography, Sephadex LH-20 column chromatography and reversed phase HPLC. Funalenone inhibited 50% of type I collagenase activity at a concentration of 170 microM, but inhibited 18.3% and 38.7% against 72 kDa and 92 kDa type IV collagenase, respectively, at a concentration of 400 microM.

A simple screening system for anti-HIV drugs: syncytium formation assay using T-cell line tropic and macrophage tropic HIV env expressing cell lines--establishment and validation.

The first step in cellular entry of HIV involves binding of the viral envelope glycoprotein complex (gp120/gp41) to specific receptor molecules on the target cells. The cell-cell fusion (syncytium formation) between env expressing cells and CD4+ cells mimics the viral infection of the host cells. To search for anti-HIV substances preventing this process, we constructed the recombinant cell lines, HeLa/CD4/Lac-Z and HeLa/T-env/Tat for T-cell tropic (HIV-1(NL4-3)) system, and HOS/CD4/CCR5/Lac-Z and HeLa/M-env/Tat for macrophage tropic (HIV-1(SF162)) system. When each pair of cells were co-incubated for 20 hours, the multinuclear giant cells (syncytia) were formed and beta-galactosidase was expressed. These systems are less biohazardous because no infectious virus particles are used. Their validity in screening for anti-HIV substances which inhibit syncytium formation was confirmed using various known HIV entry inhibitors.

Andrastins A-C, new protein farnesyltransferase inhibitors produced by Penicillium sp. FO-3929. I. Producing strain, fermentation, isolation, and biological activities.

New protein farnesyltransferase inhibitors, andrastins A-C, have been discovered in the cultured broth of Penicillium sp. FO-3929. Andrastins extracted from broth supernatant were purified by silica gel chromatography, ODS chromatography and HPLC. The IC50 of andrastins A, B, and C against protein farnesyltransferase were 24.9, 47.1, and 13.3 microM, respectively.

Phenopyrrozin, a new radical scavenger produced by Penicillium sp. FO-2047.

A new radical scavenger, named phenopyrrozin, was isolated from the culture broth of Penicillium sp. FO-2047. Phenopyrrozin was purified from whole broth solvent extraction, silica gel chromatography, and HPLC. The structure of phenopyrrozin was elucidated as 5,6,7,7a-tetrahydro-2-hydroxy-1-phenyl-3H-pyrrolizin-3-one. The IC50 of phenopyrrozin against lipid peroxidation induced by Cr2K2O7 was 73 micrograms/ml. Phenopyrrozin also reduced chromosomal aberrations induced by paraquat.

Kurasoins A and B, new protein farnesyltransferase inhibitors produced by Paecilomyces sp. FO-3684. II. Structure elucidation and total synthesis.

The structures of new protein farnesyltransferase inhibitors, kurasoins A and B, were elucidated by NMR study. Kurasoins A and B are acyloin compounds having in common a 3-hydroxy-1-phenyl-2-butanone moiety, to which p-hydroxyphenyl and 3-indolyl moieties respectively, are connected at C-4. The structures were confirmed by total synthesis.

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene.

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the alpha subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC50) of the enzyme activity was measured to be 400 microM, whereas the IC50 value was 15 microM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 phenotype and showed cerulenin resistance.(ABSTRACT TRUNCATED AT 250 WORDS)