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.

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces.

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.

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 pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase.

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.

The joining of ribosomal subunits in eukaryotes requires eIF5B.

Initiation of eukaryotic protein synthesis begins with the ribosome separated into its 40S and 60S subunits. The 40S subunit first binds eukaryotic initiation factor (eIF) 3 and an eIF2-GTP-initiator transfer RNA ternary complex. The resulting complex requires eIF1, eIF1A, eIF4A, eIF4B and eIF4F to bind to a messenger RNA and to scan to the initiation codon. eIF5 stimulates hydrolysis of eIF2-bound GTP and eIF2 is released from the 48S complex formed at the initiation codon before it is joined by a 60S subunit to form an active 80S ribosome. Here we show that hydrolysis of eIF2-bound GTP induced by eIF5 in 48S complexes is necessary but not sufficient for the subunits to join. A second factor termed eIF5B (relative molecular mass 175,000) is essential for this process. It is a homologue of the prokaryotic initiation factor IF2 (re and, like it, mediates joining of subunits and has a ribosome-dependent GTPase activity that is essential for its function.

Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide 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.

Expression of the Streptomyces alboniger pur cluster in Streptomyces lividans is dependent on the bldA-encoded tRNALeu.

Streptomyces lividans 1326-9, a bldA+ strain, and its bldA39 mutant derivative J1725 were transformed with a cosmid containing the pur cluster, which determines the puromycin biosynthetic pathway from Streptomyces alboniger. bldA+ transformants produced puromycin in typical amounts, whereas bldA39 transformants did so at drastically decreased levels. Transformation of low producers with the wild-type bldA gene reverted this phenotype to normal production. These data, in addition to the presence of a TTA codon in the amino-terminal coding region of the pur10 and pur6 genes of the pur cluster, suggest that the puromycin biosynthetic pathway is translationally dependent on the bldA gene product, a tRNALeu.

Structure/function correlation of spermine-analogue-induced modulation of peptidyltransferase activity.

In a cell-free system derived from Escherichia coli, various analogues of spermine were used to study their effect on the binding of AcPhe-tRNA to poly (U)-programmed ribosomes and on the puromycin reaction carried out at 6 mM Mg2+ (Ac, acetyl). In the absence of factors washable from ribosomes (FWR fraction), mono-acylated or di-acylated analogues of spermine stimulate the binding of AcPhe-tRNA to a lesser degree than spermine, in the order: N1-acetylspermine > N1,N12-diacetylspermine approximately = N1,N12-dipivaloylspermine. Also, the above analogues do not show any sparing effect on Mg2+ requirements for AcPhe-tRNA binding to ribosomes, in contrast to spermine. The presence of FWR fraction during the binding or acetylation of the secondary amines of spermine moderates or abolishes the stimulatory effect. In addition, all analogues tested enhance the stability of the ternary complex AcPhe-tRNA-poly(U)-ribosome and the extent of AcPhe-puromycin synthesis, particularly in the absence of the FWR fraction. At the kinetic phase of AcPhe-puromycin synthesis, the analogues display both stimulatory and inhibitory effects, depending on the absence (partial noncompetitive inhibition) or the presence of the FWR fraction (nonessential activation in concert with partial noncompetitive inhibition). Detailed kinetic analysis shows that the analogues tested can mimic the behaviour of spermine, however, the potency to affect the peptidyltransferase activity depends on their degree of acylation, acyl-substituent size, charge distribution and on their chain flexibility.

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).

Characterization of yeast translation initiation factor 1A and cloning of its essential gene.

Translation initiation factor eIF1A is required in vitro for maximal rates of protein synthesis in mammalian systems. It functions primarily by dissociating ribosomes and stabilizing 40 S preinitiation complexes. To better elucidate its precise role in promoting the translation initiation process, the yeast form of eIF1A has been identified in Saccharomyces cerevisiae and purified to homogeneity on the basis of its cross-reaction with antibodies prepared against mammalian eIF1A. The apparent mass of yeast eIF1A (22 kDa) resembles that of the mammalian homolog (20 kDa), and the yeast factor is active in stimulating methionyl-puromycin synthesis in an assay composed of mammalian components. The gene encoding yeast eIF1A, named TIF11, was cloned and shown to be single copy. TIF11 encodes a protein comprising 153 amino acids (17.4 kDa); the deduced amino acid sequence exhibits 65% identity with the sequence of human eIF1A. Both human and yeast eIF1A contain clusters of positive residues at the N terminus and negative residues at the C terminus. Deletion/disruption of TIF11 demonstrates that eIF1A is essential for cell growth. Expression of human eIF1A cDNA rescues the growth defect of TIF11-disrupted cells, indicating that the structure/function of yeast and mammalian eIF1A is highly conserved.

Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites.

The number of tRNA binding sites in 80S ribosomes from Saccharomyces cerevisiae was assessed by means of tRNA saturation and translocation experiments. In the absence of cognate mRNA yeast ribosomes could bind 0.6 [32P]tRNA(Phe) per 80S while poly(U) programmed ribosomes accepted up to 1.7 tRNA(Phe) molecules per 80S or 0.5 molecules of Ac[14C]Phe-tRNA(Phe) per 80S. Compared with the known features of E. coli ribosomes these binding values indicated both the presence of three tRNA binding sites and the validity of the exclusion principle for peptidyl-tRNA binding to yeast ribosomes. Upon EF-2 dependent translocation of a complex containing deacyl-tRNA in the P-site and AcPHe-tRNA in the A-site, the deacylated tRNA does not leave the ribosome quantitatively. This observation suggests the presence of an E site in 80S ribosomes which is functionally equivalent to the one previously characterized in prokaryotic systems.

Identification of the gene encoding an N-acetylpuromycin N-acetylhydrolase in the puromycin biosynthetic gene cluster from Streptomyces alboniger.

The biologically inactive compound N-acetylpuromycin is the last intermediate of the puromycin antibiotic biosynthetic pathway in Streptomyces alboniger. Culture filtrates from either this organism or Streptomyces lividans transformants harboring the puromycin biosynthetic gene cluster cloned in low-copy-number cosmids contained an enzymic activity which hydrolyzes N-acetylpuromycin to produce the active antibiotic. A gene encoding the deacetylase enzyme was located at one end of this cluster, subcloned in a 2.5-kb DNA fragment, and expressed from a high-copy-number plasmid in S. lividans.

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine in Saccharomyces cerevisiae.

Translation initiation factor eIF-5A (formerly called eIF-4D) is a small, highly conserved protein in eukaryotic cells that undergoes a unique modification at one of its lysine residues to form hypusine. eIF-5A stimulates in vitro the synthesis of methionyl-puromycin, a model reaction for formation of the first peptide bond. In Saccharomyces cerevisiae eIF-5A is encoded by two highly homologous genes, TIF51A and TIF51B, and each gene gives rise to two hypusinated isoelectric variants, eIF-5Aa (more acidic) and eIF-5Ab (more basic). In order to study the structural and functional differences between the two isoforms, both isoelectric forms were purified from a yeast strain overexpressing TIF51A and were shown to stimulate identically the synthesis of methionyl-puromycin in a heterologous mammalian assay system. Pulse-chase labeling of yeast cells with [35S]methionine showed that the basic form, eIF-5Ab, is a precursor form of the acidic form, eIF-5Aa. Immunoprecipitation of 32P-labeled cell lysates with rabbit antibodies specific for yeast eIF-5A, phosphoprotein phosphatase treatment of eIF-5Aa, and phosphoamino acid analysis demonstrated that eIF-5Aa is generated by phosphorylation of eIF-5Ab on serine. Therefore eIF-5A undergoes two post-translational modifications, hypusination and phosphorylation, where the activity of the factor is dependent on the first but is not influenced in vitro by the second.

Cosmid pJAR4, a novel Streptomyces-Escherichia coli shuttle vector for the cloning of Streptomyces operons.

A novel shuttle cosmid vector (pJAR4), based on pK505, was constructed for the cloning of Streptomyces DNA. It is a low-copy-number vector which determines hygromycin B-resistance as a selective marker and was used to clone the puromycin biosynthesis pathway from Streptomyces alboniger. Cosmids pJAR4 and pKC505 (which determines apramycin-resistance) stably co-transform both Streptomyces lividans and Streptomyces griseofuscus.

Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.

Comparison of the activities of variant forms of eIF-4D. The requirement for hypusine or deoxyhypusine.

Eukaryotic protein synthesis initiation factor 4D (eIF-4D) (current nomenclature, eIF-5A) contains the unique amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). The first step in hypusine biosynthesis, i.e. the formation of the intermediate, deoxyhypusine (N epsilon-(4-aminobutyl)lysine), was carried out in vitro using spermidine, deoxyhypusine synthase, and ec-eIF-4D(Lys), an eIF-4D precursor prepared by over-expression of human eIF-4D cDNA in Escherichia coli. In a parallel reaction, using N-(3-aminopropyl)cadaverine in place of spermidine, a variant form of eIF-4D containing homodeoxyhypusine (N epsilon-(5-aminopentyl)lysine) was prepared. Evidence that N-(3-aminopropyl)cadaverine can also act as the amine substrate for deoxyhypusine synthase in intact cells was obtained by incubating putrescine- and spermidine-depleted Chinese hamster ovary cells with [3H]cadaverine. In these cells, in which [3H]cadaverine is readily converted to N-(3-aminopropyl) [3H]cadaverine, small amounts of [3H]homodeoxyhypusine and another 3H-labeled compound, presumed to be N epsilon-(5-amino-2-hydroxy[3H]pentyl)lysine, were found. eIF-4D stimulates methionyl-puromycin synthesis, an in vitro model assay for translation initiation. Whereas the unmodified precursor ec-eIF-4D(Lys) appeared inactive, the deoxyhypusine-containing form provided a significant degree of stimulation. The variant form containing homodeoxyhypusine, on the other hand, showed little or no activity. These findings emphasize the importance of hypusine or deoxyhypusine for the biological activity of eIF-4D and demonstrate the influence of both the length and chemical nature of its amino alkyl side chain.

The role of mammalian initiation factor eIF-4D and its hypusine modification in translation.

Initiation factor eIF-4D functions late in the initiation pathway, apparently during formation of the first peptide bond. The factor is post-translationally modified at a specific lysine residue by reaction with spermidine and subsequent hydroxylation to form hypusine. A precursor form lacking hypusine is inactive in the assay for methionyl-puromycin synthesis, but activity is restored following in vitro modification to deoxyhypusine, thereby suggesting that the modification is essential for function. Since formylated methionyl-tRNA is less dependent on eIF-4D in the puromycin assay, we postulate that eIF-4D and its hypusine modification may stabilize charged Met-tRNA binding to the peptidyl transferase center of the 60S ribosomal subunit. Analysis of eIF-4D genes in yeast indicate that eIF-4D and its hypusine modification are essential for cell growth.