Puromycins B-E, Naturally Occurring Amino-Nucleosides Produced by the Himalayan Isolate Streptomyces sp. PU-14G.

The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2″-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2″-amine and the impact of 3'-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3'- N-amino acid substitution that differs from the 3'- N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation.

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.

The pur7 gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger encodes a nudix hydrolase.

Pur7 is the product of a gene from the puromycin biosynthetic pur cluster of Streptomyces alboniger. It was expressed in Escherichia coli as a recombinant protein fused to a His tag and then was highly purified through a Ni(2+) column. It showed a 3'-amino-3'-dATP pyrophosphohydrolase (nudix) activity which produced 3'-amino-3'-dAMP and pyrophosphate. This is consistent with the presence of a nudix box in its amino acid sequence. As observed with other nudix hydrolases, Pur7 has an alkaline pH optimum and a requirement for Mg(2+). Among a large variety of other nucleotides tested, only 3'-amino-3'-dTTP was a Pur7 substrate, although at lower reaction rates than 3'-amino-3'-dATP. These findings suggest that Pur7 has a high specificity for the 3' amino group at the ribofuranoside moiety of these two substrates. The K(m) and V(max) values for these dATP and dTTP derivatives were 120 microM and 17 microM/min and 3.45 mM and 12.5 microM/min, respectively. Since it is well known that 3'-amino-3'-dATP is a strong inhibitor of DNA-dependent RNA polymerase, whereas 3'-amino-3'-dAMP is not, Pur7 appears to be similar to other nudix enzymes in terms of being a housecleaning agent that permits puromycin biosynthesis to proceed through nontoxic intermediates. Finally, the identification of this activity has allowed a revision of the previously proposed puromycin biosynthetic pathway.

The Pur10 protein encoded in the gene cluster for puromycin biosynthesis of Streptomyces alboniger is an NAD-dependent ATP dehydrogenase.

The pur10 gene of the puromycin (pur) cluster of Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Highly purified Pur10 protein, obtained in Escherichia coli as a recombinant protein fused to a histidine tail, had an NAD-dependent ATP dehydrogenase activity. The Km and Vmax values for ATP were 0.49 mM and 14.5 nmol/min and for NAD 0.53 mM and 15.2 nmol/min, respectively. The ATP-derived product of the reaction apparently decomposed producing a triphosphorylated compound plus an adenine derivative. These and previous results suggested that Pur10 carries out the first step of the puromycin biosynthetic pathway, namely, conversion of ATP into 3'-keto-3'-deoxyATP.

New aspects on the kinetics of activation of ribosomal peptidyltransferase-catalyzed peptide bond formation by monovalent ions and spermine.

The effect of NH4+ and K+ ions on the activity of ribosomal peptidyltransferase was investigated in a model system derived from Escherichia coli, in which AcPhe-puromycin is produced by a pseudo-first-order reaction between the preformed AcPhe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin. Detailed kinetic analysis suggests that both NH4+ and K+ ions act as essential activators of peptidyltransferase by filling randomly, but not cooperatively, multiple sites on the ribosome. With respect to the NH4+ effect at 25 degrees C. the values of the molecular interaction coefficient (n), the dissociation constant (KA), and the apparent catalytic rate constant (kmax) of peptidyltransferase at saturating levels of NH4+ and puromycin are 1.99, 268.7 mM and 24.8 min(-1), respectively. The stimulation of peptidyltransferase by K+ ions at 25 degrees C (n = 4.38, KA = 95.5 mM, kmax = 9.6 min[-1]) is not as marked as that caused by NH4+ ions. Furthermore, it is evident that NH4+ at high concentration (200 mM) is effective in filling regulatory sites of complex C, which are responsible for the modulatory effect of spermine. The combination of NH4+ ions (200 mM) with spermine (300 microM) produces an additive increase in peptidyltransferase activity. Taken together, these findings suggest the involvement of two related pathways in the regulation of peptidyltransferase activity, one mediated by specific monovalent cations and the other mediated by spermine.

The biosynthetic pathway of the aminonucleoside antibiotic puromycin, as deduced from the molecular analysis of the pur cluster of Streptomyces alboniger.

The pur cluster which encodes the puromycin biosynthetic pathway from Streptomyces alboniger was subcloned as a 13-kilobase fragment in plasmid pIJ702 and expressed in an apparently regulated manner in the heterologous host Streptomyces lividans. The sequencing of a 9.1-kilobase DNA fragment completed the sequence of pur. This permitted identification of seven new open reading frames in the order: napH, pur7, pur10, pur6, pur4, pur5, and pur3. The latter is followed by the known pac, dmpM, and pur8 genes. Nine open reading frames are transcribed rightward as a unit in opposite direction to that of the pur8 gene which is expressed as a monocistronic transcript from the right-most end. napH encodes the known N-acetylpuromycin N-acetylhydrolase. The deduced products from other open reading frames present similarities to: NTP pyrophosphohydrolases (pur7), several oxidoreductases (pur10), the putative LmbC protein of the lincomycin biosynthetic pathway from Streptomyces lincolnensis (pur6), S-adenosylmethionine-dependent methyltransferases (pur5), a variety of presumed aminotransferases (pur4), and several monophosphatases (pur3). According to these similarities and to previous biochemical work, a puromycin biosynthetic pathway has been deduced. No cluster-associated regulatory gene was found. However, both pur10 and pur6 genes contain a TTA codon, which suggests that they are translationally controlled by the bldA gene product, a specific tRNA(Leu).

Two genes in Streptomyces alboniger puromycin biosynthesis pathway are closely linked.

Several recombinant plasmids, derived from the Streptomyces vector pIJ702 and carrying different stretches of Streptomyces alboniger DNA encoding the gene (pac) for puromycin N-acetyl transferase [Vara et al., Gene 33 (1985) 197-206] were found to also include the gene (dmpM) for the O-demethylpuromycin O-methyl transferase enzyme. Both genes are present on the same 2.4-kb DNA fragment. Coupled transcription-translation experiments suggested that the dmpM gene product is a 44-kDa polypeptide and that both dmpM and pac might belong to different transcriptional units. The level of expression of the dmpM gene was dependent upon the orientation of insertion in the vector.

Hepatic protein synthetic activity in vivo after ethanol administration.

Hepatic protein synthetic activity in vivo was measured by the incorporation of [3H]puromycin into elongating nascent polypeptides of rat liver to form peptidyl-[3H]puromycin. Our initial experiments showed that saturating doses of [3H]puromycin were achieved at 3-6 mumol/100 g body weight, and that maximum labeling of nascent polypeptides was obtained 30 min after injection of the labeled precursor. Labeled puromycin was found to be suitable for measuring changes in the status of protein synthesis, since the formation of the peptidyl-[3H]puromycin was decreased in fasted animals and was increased in rats pretreated with L-tryptophan. [3H]Puromycin incorporation into polypeptides was then measured after acute ethanol administration as well as after prolonged consumption of ethanol which was administered as part of a liquid diet for 31 days. Acute alcohol treatment caused no significant change in [3H]puromycin incorporation into liver polypeptides. In rats exposed to chronic ethanol feeding, peptidyl-[3H]puromycin formation, when expressed per mg of protein, was slightly lower compared to pair-fed controls, but was unchanged compared to chow-fed animals. When the data were expressed per mg of DNA or per 100 g body wt, no differences in protein synthetic activity were observed among the three groups. These findings indicate that neither acute nor chronic alcohol administration significantly affects protein synthetic activity in rat liver. They further suggest that accumulation of protein in the liver, usually seen after prolonged ethanol consumption, is apparently not reflected by an alteration of hepatic protein synthesis.

Translational initiation factors from sea urchin eggs and embryos: functional properties are highly conserved.

We have used three mammalian in vitro assays for translational initiation (globin synthesis, methionyl-puromycin synthesis, and ternary complex formation), consisting of defined components, to ask whether sea urchin (Strongylocentrotus purpuratus) egg and embryo translational components are active in heterologous assays for mammalian components, and to determine to what extent these activities are evolutionarily conserved. A "pH 5 enzyme" fraction prepared from unfertilized eggs and embryos efficiently replaced the rat liver pH 5 fraction in a globin synthesis assay, indicating that the elongation and termination factors and the aminoacyl-tRNAs are compatible with the mammalian translational machinery. The classical schemes for mammalian initiation factor purification yielded low or no detectable activities in the ribosomal salt washes, so a novel procedure was developed to partially purify initiation factors from sea urchin eggs and embryos before testing for activity. A 12,000 g homogenate from unfertilized eggs was fractionated by step elution from phosphocellulose at 100, 300, 600, and 1,200 mM salt. Initiation factor activities were found in each salt step as predicted for the mammalian counterparts. The following activities have been detected: eIF2, eIF3/4F, eIF4A, eIF4B, eIF4C, eIF4D, and eIF5. Further fractionation of each elution step yielded preparations enriched in specific initiation factor activities. However, denaturing polyacrylamide gel electrophoresis of the fractions gave complex polypeptide patterns and no clearly identifiable bands corresponding to the mammalian initiation factor polypeptides. In spite of the conservation of factor activity, crude and affinity purified polyclonal antibodies to the mammalian factors did not cross-react with the sea urchin preparations. The demonstration that initiation factor activities are sufficiently conserved to allow their being assayed is the first step in our dissection of the translational machinery of eggs and embryos, and in the complete analysis of the regulation of translation during early development.

Mechanism of self-protection in a puromycin-producing micro-organism.

Puromycin is a potent inhibitor of bacterial protein synthesis, but puromycin-producing Streptomyces alboniger KCC S-0309 is tolerant to the antibiotic in vivo. Puromycin bound to both 30S and 50S ribosomal subunits from S. alboniger and inhibited polyuridylate-directed polyphenylalanine synthesis by the ribosomes. However, the organism possessed a novel puromycin-inactivating enzyme which acetylated the antibiotic at the 2''-NH2 group of the O-methyltyrosine moiety.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis.

New evidence for the inhibition by harringtonine in the formation of the first peptide bond in protein synthesis was obtained by means of experiments in the cell-free system. The antitumour drug strongly blocked dipeptide synthesis in the system without elongation factor G and GTP. Furthermore, it inhibited N-Acetyl-phenylalanyl-puromycin formation from N-Acetyl-phenylalanyl-tRNA, puromycin, and ribosomes.

Necessity of the sulfoxide moiety for the biochemical and biological properties of an analog of sparsomycin.

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.

Control of differentiation in streptomycetes: involvement of extrachromosomal deoxyribonucleic acid and glucose repression in aerial mycelia development.

When Streptomyces alboniger spores were grown in Hickey-Tresner broth containing 5 muM ethidium bromide, a high frequency of permanently cured aerial mycelia-negative (am-) colonies was recovered. The appearance an am- colonies was time dependent: a very low frequency (0.3%) at zero time, a maximum (9 to 21%) after 2 to 5 days of growth, and a decline again to low frequencies later in the growth cycle. On agar, cured am- colonies of S. alboniger still produced puromycin. The development of aerial mycelia in S. alboniger, S. scabies, and S. coelicolor was also sensitive to glucose repression. Colonies grown on Hickey-Tresner agar containing 2% glucose remained phenotypically am- throughout the observation period. Adenine (2.5 mM or greater), and to a lesser extent adenosine and guanosine, specifically reversed the repression. The accumulation of undissociated organic acids appears to be involved in glucose repression of aerial mycelia formation. However, this does not appear to be the case with puromycin production in S. alboniger; glucose repression was observed over the pH range 5.0 to 7.5.

Biosynthesis of puromycin in Streptomyces alboniger: regulation and properties of O-demethylpuromycin O-methyltransferase.

Mechanisms for regulation of puromycin biosynthesis in Streptomyces alboniger were studied by measuring the levels of S-adenosyl-l-methionine:O-demethylpuromycin O-methyltransferase. The enzyme was released in soluble form from mycelia by 3 to 5 min of sonication at 4 C. Maximal specific activities of 0.7 and 0.1 nmol/min per mg of protein were found in cells grown in corn steep liquor-corn starch and Hickey-Tresner media, respectively. In both media, the O-methyltransferase activity rose from low levels to a maximum during midlogarithmic growth and then declined or disappeared completely (in Hickey-Tresner medium) during stationary phase. Either glucose (1%) or ethidium bromide (5 muM) reduced O-methyltransferase formation to very low levels with no effect on overall growth. Complete glucose repression of antibiotic formation occurred on agar. Cells grown in the presence of ethidium bromide continued to produce low enzyme levels after regrowth in the absence of dye, but formed normal amounts of puromycin on Hickey-Tresner agar. The O-methyltransferase, either crude or purified, was rapidly inactivated at 37 C. Each substrate alone, or both together at lower concentrations, protected against this loss of activity. Puromycin inhibited the transferase. Regulation of O-methyltransferase synthesis in S. alboniger includes (i) induction early in growth that is susceptible to catabolite repression and differential inhibition by ethidium bromide, and (ii) protection of the enzyme from inactivation by increased intracellular levels of its substrates. The O-methyltransferase was purified 30- to 40-fold by a combination of protamine sulfate precipitation, ammonium sulfate fractionation, adsorption and gradient salt elution from diethylaminoethyl-cellulose and Sephadex G-200 gel filtration. The enzyme was very unstable, even at low temperatures, upon purification beyond the salt fractionation step, but was stabilized by the addition of S-adenosyl-l-methionine during later stages of purification.

Reversal of pactamycin inhibition of methionyl-puromycin synthesis and 80S initiation complex formation by a ribosomal joining factor.

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA(f) to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type. In the absence of the factor, [(35)S]Met-puromycin was not synthesized and [(35)S]Met-tRNA(f) bound only to the small ribosomal subunit. The amount of [(35)S]Met-tRNA(f) bound to 80S ribosomes bearing endogenous mRNA and the amount of [(35)S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a "joining factor," and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.

Protein synthesis in Bacillus subtilis: differential effect of potassium ions on in vitro peptide chain initiation and elongation.

The preparation and fractionation of a highly active and stable in vitro protein-synthesizing system from Bacillus subtilis is described. Potassium satisfied the requirement for a monovalent ion when the initiation factor-dependent binding of formyl-methionyl-transfer ribonucleic acid and synthesis of formyl-methionyl-puromycin were assayed, whereas it inhibited the reactions for polyphenylalanine synthesis. On the other hand, the ammonium ion satisfied the requirement for all assayed reactions. The in vitro experimental evidence suggested that potassium is an inhibitor of one or a few specific reactions involved in peptide chain elongation in B. subtilis.