Rapid typing and elucidation of new secondary metabolites of intact cyanobacteria using MALDI-TOF mass spectrometry.

Toxic cyanobacterial blooms are a threat because of secondary metabolite production. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to identify intact microorganisms. Microgram quantities of prepared cells, including solvent (acetonitrile and ethanol) and alpha-cyano-4-hydroxycinnamic acid matrix, display spectra showing predominantly the secondary metabolites including known microcystins, micropeptin, and anabaenopeptolin. A new cyclic anabaenopeptolin has been identified using the Post-Source-Decay mode. Strains of various origins can easily be typed according to their cyclic peptide production, and toxic and nontoxic algal blooms can be differentiated within minutes.

Relationship between activating and editing functions of the adenylation domain of apo-tyrocidin synthetase 1 (apo-TY1).

Tyrocidine synthetase 1 (TY1), the initial monomodular constituent of the tyrocidine biosynthetic system, exhibits relaxed substrate specificity, however an efficient editing of the mis-activated amino acid provides for fidelity of product formation. We chose to analyse the consequence of single amino acid substitutions, in the amino acid activation site of apo-TY1, on the editing functions of the enzyme. Discrimination between L-Phe and D-Phe by apo-TY1 depends primarily on the editing reaction. Distraction of unnatural amino acid substrates, such as L-PheSer, implies that editing is not designated to select a specific mis-activated amino acid, but instead to discriminate all mis-activated amino acid analogues. It was shown that active site residues which interact with the adenylate are essential for both activation and editing. Substitution of Lys186 with arginine substantially reduces the editing capacity of the protein. Loss of amino acid discrimination ability by the apo-K186T and apo-R416T mutant proteins suggests a role of active site residues in maintaining the structural determinants for substrate selection. Inadequate conformational changes, induced by non-cognate amino acid substrates, promote ATP breakdown yielding P(i) and ADP. Replacement of residue Lys186 or Arg416 enhances ATP hydrolysis implying a role in binding or adjusting of the triphosphate chain for adenylate formation and pyrophosphate cleavage.

Synthesis of (di)adenosine polyphosphates by non-ribosomal peptide synthetases (NRPS).

In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase. The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase. The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule. In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively. A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.

Probing the domain structure and ligand-induced conformational changes by limited proteolysis of tyrocidine synthetase 1.

The boundaries of the structural domains in peptide synthetases and the conformational changes related to catalysis were investigated by limited proteolysis of tyrocidine synthetase 1 (TY1). Four regions sensitive to proteolysis were detected (cleavage site at Arg13, Arg424, Arg509 and Arg602) that, in addition to an N-terminal extension, accurately delineate the domain boundaries of the adenylate-forming domain, the aminoacyl carrier domain, and the epimerisation domain. Limited proteolysis of an active N-terminal truncated deletion mutant, His6DeltaTY1, generated two stable and structurally independent subunits, corresponding to the subdomains of the adenylation domain. The structural integrity of the carrier domain was substantiated by its resistance to proteolytic degradation. Evidence is provided that the C-terminal "spacer" region with epimerising and/or condensing activity folds into an autonomous domain stable against degradation by limited proteoly sis. In the presence of substrates, reduced susceptibility to proteolysis was observed in the linker region connecting the subdomains of the adenylation domain, and corresponding to a peptide stretch of low electron density in the X-ray structure of the homologous firefly luciferase. Sequence analysis has shown that the respective linker contains conserved residues, whereas the linker regions connecting the structural domains are of low homology with a significant content of Pro, Ala, Glu and polar residues. A combination of kinetic and proteolytic studies using ATP analogues with substitutions in the phosphate chain, AMP-PcP, AMP-PNP and AMP-cPP, strongly suggests that the generation of a productive complex is associated with the ability of the beta, gamma-pyrophosphate moiety of ATP to adopt the proper active-site conformation. These data substantiate the observation that peptide synthetases undergo a series of conformational changes in the process of adenylate formation and product release.

Thioesterase domain of delta-(l-alpha-Aminoadipyl)-l-cysteinyl-d-valine synthetase: alteration of stereospecificity by site-directed mutagenesis.

The carboxy-terminal thioesterase domain of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase catalyzes the hydrolytic release of the tripeptide product (LLD-ACV). By site-directed mutagenesis an S3599A change was introduced into the highly conserved GXSXG motif, resulting in a more than 95 % decrease of penicillin production. Purification of the modified multienzyme showed surprisingly only a 50 % reduction of the peptide formation rate, with the stereoisomer delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine (LLL-ACV) as the dominating product. Thioesterases of ACV synthetases differ from other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXSXG-seryl residue is apparently not essential in acyl transfer leading to peptide release. Instead, this motif may be involved in the control of tripeptide epimerization by selection of the isomer to be released, and the construct supports the presence of LLL-ACV as an intermediate in penicillin biosynthesis.

The nonribosomal code.

How genes are expressed and translated into proteins (using mRNA, codons and tRNAs as adaptor molecules) forms the basis of the 'genetic code'. Many peptides are synthesized nonribosomally, however, by large protein complexes that also serve as templates. Recent advances have shed light on what the nonribosomal code is and how it can be read.

Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide-polyketide synthetase system.

BACKGROUND: Blooms of toxic cyanobacteria (blue-green algae) have become increasingly common in the surface waters of the world. Of the known toxins produced by cyanobacteria, the microcystins are the most significant threat to human and animal health. These cyclic peptides are potent inhibitors of eukaryotic protein phosphatases type 1 and 2A. Synthesized nonribosomally, the microcystins contain a number of unusual amino acid residues including the beta-amino polyketide moiety Adda (3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyl-4,6-decadienoic acid). We have characterized the microcystin biosynthetic gene cluster from Microcystis aeruginosa PCC7806. RESULTS: A cluster spanning 55 kb, composed of 10 bidirectionally transcribed open reading frames arranged in two putative operons (mcyA-C and mcyD-J), has been correlated with microcystin formation by gene disruption and mutant analysis. Of the 48 sequential catalytic reactions involved in microcystin synthesis, 45 have been assigned to catalytic domains within six large multienzyme synthases/synthetases (McyA-E, G), which incorporate the precursors phenylacetate, malonyl-CoA, S-adenosyl-L-methionine, glutamate, serine, alanine, leucine, D-methyl-isoaspartate, and arginine. The additional four monofunctional proteins are putatively involved in O-methylation (McyJ), epimerization (McyF), dehydration (McyI), and localization (McyH). The unusual polyketide amino acid Adda is formed by transamination of a polyketide precursor as enzyme-bound intermediate, and not released during the process. CONCLUSIONS: This report is the first complete description of the biosynthesis pathway of a complex cyanobacterial metabolite. The enzymatic organization of the microcystin assembly represents an integrated polyketide-peptide biosynthetic pathway with a number of unusual structural and enzymatic features. These include the integrated synthesis of a beta-amino-pentaketide precursor and the formation of beta- and gamma-carboxyl-peptide bonds, respectively. Other features of this complex system also observed in diverse related biosynthetic clusters are integrated C- and N-methyltransferases, an integrated aminotransferase, and an associated O-methyltransferase and a racemase acting on acidic amino acids.

Antibiotics--cloning of biosynthetic pathways.

Biosynthetic pathways leading to antibiotics have often been found to be clustered, and new organizational forms of multifunctional enzymes have been discovered. Such polyenzymes accomplish the synthesis of complex metabolites such as peptides or polyketides by a sequence of enzymatic reactions. So, reactions leading to the tripeptide precursor of beta-lactam antibiotics, ACV, or to the cycloundecapeptide cyclosporine have been fused into single polypeptide chain synthetases, respectively. In certain isofunctional sites restricted similarities have been detected.

Active site titration of gramicidin S synthetase 2: evidence for misactivation and editing in non-ribosomal peptide biosynthesis.

The catalytic competence of gramicidin S synthetase 2 (GS2) was determined by following the kinetics of PP(i) generation using active site titration measurements with [gamma-(32)P]ATP. The initial 'burst' of product formation can be correlated to the generation of the aminoacyl adenylate:enzyme complexes at the four amino acid activation domains and the subsequent aminoacylation of carrier domains, followed by a slow linear turnover of substrate due to breakdown of the intermediate. Simultaneous activation of all four amino acid substrates at a saturating concentration displayed a consumption of 8.3 ATP/GS2. In the presence of single amino acids, a binding stoichiometry higher than the anticipated two ATP per active site was obtained, implying misactivation at non-cognate domains. Breakdown of acyladenylate intermediates reflects a possible corrective mechanism by which the enzyme controls the fidelity of product formation.

Penicillin biosynthesis: intermediates of biosynthesis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine formed by ACV synthetase from Acremonium chrysogenum.

The tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) is synthesised by the multifunctional enzyme ACV synthetase integrating four steps of the penicillin and cephalosporin biosynthetic pathway. Peptide synthesis follows the thiotemplate mechanism from intermediates bound as thioesters to the enzyme. The formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-thioester in the absence of L-valine was shown by isolation of the enzyme-substrate complex and cleavage of the covalently bound intermediate with performic acid. The dipeptide was recovered as cysteic acid or cysteic acid oxime and detected by HPLC and MALDI-TOF mass spectrometry. We conclude that the first peptide bond is formed between delta-carboxyl of L-aminoadipic acid and L-cysteine, followed by addition of the dipeptidyl intermediate to L-valine.

Dipeptide synthesis by an isolated adenylate-forming domain of non-ribosomal peptide synthetases (NRPS).

A deletion mutant of tyrocidine synthetase 1 (DeltaDeltaTY1), comprising the adenylation domain of TY1 as an independent functional adenylate-forming unit, was used to investigate the ability of the adenylation domain in non-ribosomal peptide synthetases to catalyse peptide bond formation from the aminoacyl adenylate intermediate. The results demonstrate that only one substrate amino acid needs to be activated as an aminoacyl adenylate. In view of the potential exploitation of peptide synthetases for enzymatic synthesis of dipeptides of choice, it is important to note that this does not necessarily require a dimodular construct or an intermediate acyl transfer step.

Expression of an active adenylate-forming domain of peptide synthetases corresponding to acyl-CoA-synthetases.

Peptide synthetases and acyl-CoA-synthetases form acyl adenylates which are transferred to CoA or enzyme-bound pantetheine. To verify the existence of an adenylate domain in peptide synthetases, a 60.8 kDa fragment of tyrocidine 1-synthetase was constructed by a 1,629 bp deletion, expressed in Escherichia coli, and characterized. The truncated multienzyme activated phenylalanine and substrate analogues with comparable kinetics as the over-expressed synthetase, as judged by ATP-[32P]PP(i) exchange reaction. Thus the N-terminal domain resembling an acyl-CoA-synthetase is an autonomous structural element. This N-terminal domain is followed by a cofactor binding domain, resembling acyl carrier proteins involved in polyketide formation.

Applications of peptide synthetases in the synthesis of peptide analogues.

Enzymatically formed peptides show positional variations as well as highly conserved amino acids. In the cases of gramicidin S, tyrocidine, linear gramicidins, enniatins, echinocandins and viridogrisein in vivo and in vitro studies indicate substrate selection at the level of amino acid activation as a major control step. Evidence for proof-reading steps beyond activation has been obtained in penicillin and cyclosporin biosynthesis. Activated substrate analogues may promote the formation of side products such as dipeptides and cyclodipeptides. Modifications of intermediates, such as N-methylation, influence the rates of peptide synthesis. These control steps pose limitations for the application of such enzyme systems in the production of peptide libraries. They may originate from a target oriented evolution of these synthetases.

Rapid screening and dereplication of bacterial isolates from marine sponges of the sula ridge by intact-cell-MALDI-TOF mass spectrometry (ICM-MS).

Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g. in unknown marine environments where large numbers of strains have to be isolated on different growth media. Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp. 1, Phakellia ventilabrum and Plakortis sp. growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast. We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates. Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis. A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas. The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization.

Peptides.

If we include beta-lactam antibiotics on the grounds that they have the same biosynthetic origin, peptides remain commercially the most important group of pharmaceuticals. However, our increasing knowledge of the genetic and enzymic background to biosynthesis, and of the regulation of metabolite production, will eventually bring a more unified approach to bioactive compounds. Mixing of structural types will become important, and we will be able to use our knowledge of biosynthetic genes and their regulatory networks. We will also benefit from an appreciation of the modular organization of catalytic functions, substrate transfer mechanisms and signalling between interacting enzymes. Since all of this is, in fact, the basis for enzymic synthesis of complex natural products in vivo, the exploitation of living cells requires mastery of a formidable network of cellular controls and compartments. For the present we are able to see fascinating connections emerging between genes in a variety of reaction sequences, not only in biosynthetic but also in degradative pathways. Peptide synthetases show surprising similarities to acylcoenzyme A synthetases, which are key enzymes in forming polyketides as well as in generating the CoA-derivatives that serve as substrates in degradative pathways. 4'-Phosphopantetheine, the functional half of CoA, plays a key role as the intrinsic transfer cofactor in various multienzyme systems. The comparatively small catalogue of reactions modifying natural products, notably epimerization, methylation, hydroxylation, decarboxylation (of peptides) and reduction/dehydration (of polyketides) can be found within or amongst biosynthetic proteins, generally as modules and organized in a specified order. The biochemist is coming close to the synthetic chemist's recipes, and may soon be recruiting proteins to carry them out.

Peptide antibiotics, beta-lactams, and related compounds.

In the field of natural peptides, beta-lactams, and related compounds, recent exciting developments are discussed. The increasing interest in this class of bioactive amino-acid derived structures has been attributed to the use of new directed screens (enzyme inhibition assays, beta-lactam detection, immunomodulator studies), new and improved applications (antibiotic, transplantation, and cancer chemotherapy), and advances in functional studies (DNA binding peptides, nucleotide complexones, cell wall and protein processing inhibitors). Peptides offer unique access to modifications and analog production by in vivo (directed biosynthesis) and in vitro procedures (enzymatic synthesis) due to their general linear precursors permitting point replacements. Of special interest are recent developments in the genetics of these compounds (cyclic peptides and beta-lactams), which will find applications in production methods in the near future.