Characterization and location of the L-proline activating fragment from the multifunctional gramicidin S synthetase 2

Gramicidin S synthetase 2 (GS2) derived from Bacillus brevis is a multifunctional single polypeptide (Mr 280,000) with a 4'-phosphopantetheine residue covalently bound to the enzyme. When GS2 was treated with trypsin or chymotrypsin, fragments with some activity were liberated. The molecular mass of the L-proline activating fragment was 114 kDa on SDS-PAGE. This fragment, when incubated with gramicidin S synthetase 1 (GS1) in the presence of phenylalanine and proline, produced D-Phe-L-Pro dipeptide. The fragment accepted D-phenylalanine from GS1 in the absence of L-proline. The L-proline activating fragment was shown to lack pantothenic acid by microbiological assay. On the other hand, the L-leucine activating fragment, which was partially purified, contained a large amount of pantothenic acid, although it did not form the D-Phe-L-Pro dipeptide. These results indicate that the L-proline activating site is located near an acceptor site for D-phenylalanine on GS2, but that it is not adjacent to a 4'-phosphopantetheine group. The N-terminal sequence (15 amino acid residues) of the L-proline activating fragment obtained by trypsin treatment was identical with that of GS2, indicating that the L-proline activating site is located at the N-terminus of the native synthetase. The N-terminal sequence of GS2 has been matched with the amino acid sequence deduced from the nucleotide sequence 71 bp downstream of the stop codon of the GS1 gene except that the first initiator methionine was not detected.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Organization of the biosynthesis genes for the peptide antibiotic gramicidin S.

A recombinant bacteriophage containing the intact Bacillus brevis gene for gramicidin S synthetase 1, grsA, and the 5' end of the gramicidin S synthetase 2 gene, grsB, was identified by screening an EMBL3 library with anti-GrsA antibodies. This clone, EMBL315, has a 14-kilobase (kb) insert that hybridizes to the previously isolated 3.9-kb fragment of the grsB gene, which encodes the 155-kilodalton ornithine-activating domain of gramicidin S synthetase 2. Deletion and subcloning experiments with the 14-kb insert located the grsA structural gene and its putative promoter on a 4.5-kb PvuII fragment which encoded the full-length 120-kilodalton protein in Escherichia coli. In addition, hybridization analysis revealed that the 5' end of the grsB gene is located approximately 3 kb from the grsA structural gene. Furthermore, these studies indicated that grsA and grsB are transcribed in opposite orientations.     

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.     

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

The proline-activating activity of the multienzyme gramicidin S synthetase 2 can be recovered on a 115-kDa tryptic fragment

The multienzyme gramicidin S synthetase 2 was treated with trypsin to obtain fragments capable of activating proline. Three different active fragments were detected. The course of proteolysis was simulated by using a concentration range of trypsin; the cleavage pattern indicated that one of the fragments was particularly stable. This fragment was purified and shown to have a molecular mass of 115 kDa. It was compared chromatographically, by SDS/PAGE, and enzymatically to a Pro-activating fragment produced by a gramicidin-S-negative mutant. It can be concluded that the proteolytic fragment represents a structure which is contained on a continuous part of the polypeptide chain of gramicidin S synthetase 2 and has a relatively compact structure. This provides evidence that the multienzyme gramicidin S synthetase 2 is, at least in part, constructed from functional domains. An approach towards extending these studies to other parts of the gramicidin S synthetase 2 molecule has also been devised. This work complements recombinant DNA studies in the area, providing stable functional fragments.     

Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone

The reactive thioester complexes of gramicidin S synthetase with substrate amino acids and intermediate peptides are slowly hydrolyzed in neutral buffer solutions under mild conditions. Fully active enzyme is recovered. These processes are strongly accelerated by certain thiol protective agents. In the presence of 1 mM dithioerythritol the half-life times of these hydrolysis reactions are in the range of 1-90 h at 3 degrees C. The thioester complex of gramicidin S synthetase 2 (GS2, the heavy enzyme) with the tripeptide DPhe-Pro-Val is distinguished by the highest stability of all these intermediates. A different decomposition pattern is observed for the thioester complex of GS2 with LOrn. Here 3-amino-2-piperidone (cyclo-LOrn) is formed in a rapid cyclization reaction. This product specifically blocks the activation center of GS2 for LOrn at the thioester binding site. All other activation reactions of gramicidin S synthetase are unaffected. A procedure for a specific labelling of the reaction centers of the multienzyme is outlined.     

Reaction mechanism of gramicidin S synthetase 1, phenylalanine racemase, of Bacillus brevis

We have demonstrated that gramicidin S synthetase 1 (GS 1), phenylalanine racemase [EC 5.1.1.11], of Bacillus brevis catalyzes the exchange between a proton in the medium and alpha-hydrogen of phenylalanine in the course of the racemase reaction by using tritiated water or L-phenyl[2,3-3H]alanine. GS 1 from some gramicidin S non-producing mutants of B. brevis lacking phenylalanine racemase activity did not catalyze the tritium exchange reaction. The proton exchange between phenylalanine bound as thioester on the GS 1-phenylalanine complex and water in the medium was detected, but 5,5'-dithiobis(2-nitrobenzoic acid)-modified complex lacked both the proton exchange and phenylalanine racemase activity. It is suggested that a base group, probably a sulfhydryl group, on the enzyme functions as proton donor and acceptor during the phenylalanine racemase reaction.     

On the domain construction of the multienzyme gramicidin S synthetase 2. Isolation of domains activating valine and leucine

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multienzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.     

Overexpression of a Streptomyces viridochromogenes gene (glnII) encoding a glutamine synthetase similar to those of eucaryotes confers resistance against the antibiotic phosphinothricyl-alanyl-alanine.

Phosphinothricyl-alanyl-alanine (PTT), also known as bialaphos, contains phosphinothricin, a potent inhibitor of glutamine synthetase (GS). A 2.75-kilobase NcoI fragment of the Streptomyces viridochromogenes PTT-resistant mutant ES2 cloned on a multicopy vector mediated PTT resistance to S. lividans and to S. viridochromogenes. Nucleotide sequence analysis of the 2.75-kb NcoI fragment revealed the presence of three open reading frames. Open reading frame 3 was termed glnII since significant similarity was found between its deduced amino acid sequence and those from GS of eucaryotes and GSII of members of the family Rhizobiaceae. Subcloning experiments showed that PTT resistance is mediated by overexpression of glnII encoding a 37.3-kilodalton protein of 343 amino acids. A three- to fourfold increase in gamma-glutamyltransferase activity could be observed in S. lividans transformants carrying the glnII gene on a multicopy plasmid. For S. viridochromogenes it was shown that PTT resistance conferred by the 2.75-kb NcoI fragment was dependent on its multicopy state. GS activity encoded by glnII was found to be heat labile. Southern hybridization with seven different Streptomyces strains suggested that they all carry two types of GS genes, glnA and glnII.     

Regulation of nitrogenase activity in Anabaena variabilis by modification of the Fe protein

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.     

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.     

Cloning of the tyrocidine synthetase 1 gene from Bacillus brevis and its expression in Escherichia coli

Summary The entire structural gene for tyrocidine synthetase 1 from Bacillus brevis ATCC 8185 has been cloned and expressed in Escherichia coli. Transformed E. coli cells were screened for their ability to produce tyrocidine synthetase 1 by in situ immunoassay using antibodies against gramicidin S synthetase 2 which cross-react with tyrocidine synthetase 1. The cloned gene is within a 5.2 kb fragment of B. brevis genomic DNA and requires no external promoter for its expression in E. coli. It was also observed that cloning of the 5.2 kb insert in the opposite orientation still resulted in a high level of tyrocidine synthetase 1 expression in transformed E. coli cells. In addition, protein blotting and partial purification of the gene product by gel filtration revealed a major protein of molecular weight about 100,000 with specific d-phenylalanine dependent ATP-³²PP_i and 2deoxy ATP-³²PP_i exchange activities. These unique activities of tyrocidine synthetase 1 were not detected in protein extracts of E. coli strains carrying the vector.     

Gramicidin S synthetase. Temperature dependence and thermodynamic parameters of substrate amino acid activation reactions

In the biosynthesis of the cyclic decapeptide antibiotic gramicidin S, the constituent amino acids are activated by a two-step mechanism involving aminoacyl adenylate and thio ester formation which are both reversible processes. The dissociation constants (KD) for the gramicidin S synthetase-substrate amino acid-thio ester complexes are 100-1000-fold lower compared to the KM data of the preceding aminoacyl adenylate reactions. The affinity for these substrates is appreciably higher at the thio template sites than at the aminoacyl adenylate reaction centers. Therefore, the activation equilibria are quantitatively shifted toward thio ester formation. A set of thermodynamic parameters for the activation processes was determined from the temperature dependence of the KM and KD data. Reaction enthalpies were obtained from a van't Hoff analysis of these constants. delta G degree for the substrate activation reactions of the heavy enzyme of gramicidin S synthetase (GS 2) is predominantly controlled by entropy contributions. In contrast, the overall activation and concomitant racemization of phenylalanine by phenylalanine racemase (GS 1) are exothermic processes which are distinguished by a small negative reaction entropy.     

Role of F1F0-ATPase in the growth of streptococcus mutans GS5

The role of F1F0-ATPase in Streptococcus mutans GS5 was investigated by isolating a mutant (NTS1) defective in enzyme activity by homologous recombination with a plasmid encoding the 5' terminal fragment of the F1F0-ATPase beta-subunit gene. The ATPase activity of NTS1 membranes was 49% that of GS5 membranes. The lag phase of the growth curve of NTS1 was longer than that for GS5, and the lag phase of GS5 and NTS1 were prolonged by the addition of ionophore gramicidin D; at stationary phase, the turbidity of the NTS1 culture was less than that of the GS5 culture. These results suggest that S. mutans F1F0-ATPase contributes to the generation of a stoichiometric electrogenic gradient effectively in the lag phase.     

An active serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase.

The condensing peptide forming multienzyme of gramicidin S synthetase (gramicidin S synthetase 2) was specifically labeled at its putative thiotemplate sites for L-valine and L-leucine by covalent incorporation of the 14C-labeled substrate amino acids. The thioester complexes of the multienzyme were digested with CNBr, Staphylococcus aureus V8 protease, and pepsin. Reaction center peptides containing the [14C]valine and [14C]leucine labels were isolated in pure form. They show a high degree of sequence similarity and contain the same consensus sequence LGGH/DXL. The labels were eliminated in the first Edman degradation step. A dehydroalanine was identified which can originate from either a cysteine or a serine. The comparison of the chemical results with the deduced amino acid sequence of the grsB gene encoding the gramicidin S synthetase 2 revealed that 4 such motifs are located within the gene structure, each of them being localized in the 3'-terminal region of one of 4 gene segments grsB1-B4. They have a size of approximately 2 kilobases and presumably code for the 4 amino acid activating domains of the synthetase. Surprisingly a serine was found at each putative substrate amino acid-binding position instead of a cysteine as postulated by the thiotemplate mechanism. Therefore the data suggest that active serine residues are involved in nonribosomal peptide syntheses of microbial peptides.