Computational Analysis of the First Biheterocyclization Site of the Antibiotic Microcin B17

Abstract

Microcin B17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2—connected bihete-rocyclic functionalities. In this study, the hexapeptide sequence surrounding the first bihete- rocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.     

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase–DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial–bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.     

The leader peptide is essential for the post-translational modification of the DNA-gyrase inhibitor microcin B17

Microcin B17 (MccB17) is a ribosomally encoded DNA-gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA , is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17–β-galactosidase fusion lacking the leader are post-translationally modified.     

Expansion of ribosomally produced natural products: a nitrile hydratase- and Nif11-related precursor family

Background  

A new family of natural products has been described in which cysteine, serine and threonine from ribosomally-produced peptides are converted to thiazoles, oxazoles and methyloxazoles, respectively. These metabolites and their biosynthetic gene clusters are now referred to as thiazole/oxazole-modified microcins (TOMM). As exemplified by microcin B17 and streptolysin S, TOMM precursors contain an N-terminal leader sequence and C-terminal core peptide. The leader sequence contains binding sites for the posttranslational modifying enzymes which subsequently act upon the core peptide. TOMM peptides are small and highly variable, frequently missed by gene-finders and occasionally situated far from the thiazole/oxazole forming genes. Thus, locating a substrate for a particular TOMM pathway can be a challenging endeavor.     

Posttranslational heterocyclization of cysteine and serine residues in the antibiotic microcin B17: distributivity and directionality

To produce the antibiotic Microcin B17, four Cys and four Ser residues are converted into four thiazoles and four oxazoles by the three subunit Microcin B17 synthetase. High-resolution mass spectrometry (MS) was used to monitor the kinetics of posttranslational heterocyclic ring formation (-20 Da per ring) and demonstrated the accumulation of all intermediates, from one to seven rings, indicating distributive processing. All of the intermediates could be converted by the enzyme to the eight ring product. Enzymatic chemoselectivity (Cys vs Ser cyclization rates) was assessed using iodoacetamido-salicylate to alkylate unreacted cysteines (+193 Da) in the 8 kDa biosynthetic intermediates; three of the first four rings formed were thiazoles, and by the five ring stage, all four of the cysteines had been heterocyclized while three of the original four serines remained uncyclized. Finally, tandem MS using a 9.4 T Fourier transform instrument with electrospray ionization was used to elaborate the major processing pathway: the first two rings formed are at the most amino proximal sites (Cys(41) then Ser(40)) followed by the remaining three cysteines at positions 48, 51, and 55. The cyclization of serines at positions 56, 62, and 65 then follows, with Ser(62) and Ser(65) the last to heterocyclize and the first of these at a slower rate. Thus, despite free dissociation of intermediates after each of seven ring-forming catalytic cycles, there is an overall directionality of ring formation from N-terminal to C-terminal sites. This remarkable regioselectivity is determined more by the substrate than the enzyme, due to a combination of (1) initial high-affinity binding of the posttranslational catalyst to the N-terminal propeptide of substrate 88mer, and (2) a chemoselectivity for thiazole over oxazole formation. This mechanism is consistent with antibiotic biosynthesis in vivo, yielding microcin with six, seven, and eight rings, all with bioactivity.     

The action of the bacterial toxin, microcin B17, on DNA gyrase

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.     

天然产物中五元杂环生物合成过程的研究进展

五元杂环是指一类含有除碳以外的杂原子的五元环有机化合物,可分为含有一个杂原子的杂环和含有两个杂原子的五元杂环。杂原子通常为氮原子、氧原子或硫原子,在成环时符合休克尔(Hückel)规则,具有芳香性。五元杂环以独特的生物学特性和参与氢键相互作用的能力,成为天然化合物发挥活性功能的重要基团。五元杂环在不同的生物合成途径中有多种环化过程,其前体可以是天然氨基酸、乙酸及乙酰辅酶A等多种形式。将从吡咯、恶唑和噻唑三种五元杂环入手,以代表性化合物为例,对其在生命体中生物合成过程的成环过程进行探讨,为组合生物合成和未知化合物中五元杂环成环过程的研究提供参考。     

Influence of aeration and carbon source on production of microcin B17 by Escherichia coli ZK650

Previous studies [Connell et al. (1987) Mol Microbiol 1: 195–201] have shown that expression of the microcin B17 (MccB17) promoter is inversely related to the growth rate of the culture, when slower growth was brought about by limitation of sources of carbon, nitrogen or phosphorus. When we used oxygen limitation to decrease growth in a glucose-based chemically defined medium, we found specific MccB17 production to be positively related to growth rate and extent. On the other hand, when we examined various nutritional variations of media, specific production of MccB17 showed a negative relationship to growth rate and extent, as would be predicted by the findings of Connell et al. (1987). Glucose, glycerol and acetate were found to repress MccB17 production; succinate was not repressive. Succinate is an excellent carbon source for production of MccB17 since high levels can be used with no or little interference in product synthesis. Received: 26 November 1996 / Accepted: 7 December 1996     

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

The 39‐kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260‐residue C‐terminal region of MccB is homologous to ubiquitin‐like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB‐catalysed C‐terminal MccA‐acyl‐adenylation is reminiscent of the E1‐catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C‐terminal di‐glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C‐terminal Asn. Furthermore, after an intramolecular rearrangement of MccA‐acyl‐adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate‐linked analogue of acyl‐adenylated aspartic acid. We report six‐crystal structures of MccB in apo, substrate‐, intermediate‐, and inhibitor‐bound forms. Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.     

Smart peptide libraries are accessible via enzymatic modifications

Summary The minireview summarizes the recent preparation of the following unusually modified combinatorial peptide collections useful for diagnostics and screening in drug finding. Tissue transglutaminase catalyzes cross couplings with transamidation between Gln and Lys peptide chains resulting in libraries with isopeptide bonds. The enzyme is involved in the triggering of autoantigenic B- and T-cell epitopes of coeliac disease. The microbial enzyme EpiD involved in lantibiotic biosynthesis catalyzes oxidative decarboxylation of C-terminal cysteine residues in peptide libraries transforming peptidyl-cysteines to peptide (2-mercaptovinyl)amides. Novel backbone modified peptide libraries are prepared using oxazole and thiazole building blocks carrying amino acid side chains. These amino acids have been found in many biologically active natural products from marine and microbial organisms such as microcin B17. Dityrosine and isodityrosine linked peptide dimer libraries are accessible by oxidative phenol coupling using horseradish peroxidase. Such structural elements are found for example in the polycyclic glycopeptide antibiotics of the vancomycin type. Microstructured layers of linear and cyclic peptide libraries are generated on transducer surfaces for cellular assays, sensor developments and even chiral recognition. Examples include a light-directed and microstructured electrochemical polymerization of phenol labelled peptides.     

Effect of simulated microgravity and shear stress on microcin B17 production by Escherichia coli and on its excretion into the medium.

Production of the antibacterial polypeptide microcin B17 (MccB17) by Escherichia coli ZK650 was inhibited by simulated microgravity. The site of MccB17 accumulation was found to be different, depending on whether the organism was grown in shaking flasks or in rotating bioreactors designed to establish a simulated microgravity environment. In flasks, the accumulation was cellular, but in the reactors, virtually all the microcin was found in the medium. The change from a cellular site to an extracellular one was apparently not a function of gravity, since extracellular production occurred in these bioreactors, irrespective of whether they were operated in the simulated microgravity or normal gravity mode. More probably, excretion is due to the much lower degree of shear stress in the bioreactors. Addition of even a single glass bead to the 50-ml medium volume in the bioreactor created enough shear to change the site of MccB17 accumulation from the medium to the cells.     

Synthesis of thiazole, imidazole and oxazole containing amino acids for peptide backbone modification.

Novel 5-ring heterocyclic building blocks are synthesized. These can be incorporated into analogs of peptide antibiotics such as microcin B17, which is a potent DNA-gyrase inhibitor that exhibits eight thiazole and oxazole moieties. In particular, the syntheses of imidazole and bisoxazole amino acids as novel peptidomimetics are reported, this includes a new procedure for the oxidative conversion of the intermediates oxazoline, imidazoline as well as oxazole-oxazoline into the corresponding heteroaromatic compounds. A mixture of 1,8-diazabicyclo-[5.4.0.]-undec-7-ene carbon tetrachloride/acetonitrile and pyridine proved to be a very effective and mild agent.     

The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase

The maturation pathway of microcin B17 (MccB17), a ribosomally synthesized peptide antibiotic which inhibits DNA gyrase, has been characterized. Synthesis of MccB17 involves several steps beginning with the translation of the MccB17 structural gene, mcbA, to yield a 69 amino acid precursor, preMccB17. PreMccB17 is then modified and folded by the action of three gene products, McbBCD, to yield proMccB17. Mutations in mcbA were isolated that permit modifications of the resulting mutant peptides, but prevent folding, suggesting that modification and folding are sequential steps. ProMccB17 is subsequently converted to MccB17 by removal of the W-terminal 26-amino-acid leader by a chromosomally encoded protease. Removal of the leader resulted in aggregation of the peptide, suggesting that the leader may function to maintain peptide solubility during synthesis in the cell. Finally, polyclonal antibodies raised against MccB17 recognize both MccB17 and proMccB17, but do not recognize preMccB17. This demonstrates the dramatic structural changes that result from the modifications and has been used to distinguish intermediates in the steps of maturation.     

The export of the DNA replication inhibitor Microcin B17 provides immunity for the host cell.

Microcin B17 (MccB17) is a peptide antibiotic which inhibits DNA replication in Enterobacteriaceae. Microcin-producing strains are immune to the action of the microcin. Physical and genetic studies showed that immunity is mediated by three genes: mcbE, mcbF and mcbG. We sequenced these genes and identified polypeptide products for mcbF and mcbG. By studying the contribution of each gene to the expression of immunity we found that immunity is determined by two different mechanisms. One of these, encoded by mcbE and mcbF, is also involved in the production of extracellular MccB17. To reconcile these observations we propose that McbE and McbF serve as a 'pump' for the export of active MccB17 from the cytoplasm. This model is supported by the predicted properties of the McbE and McbF proteins, which are thought to be, respectively, an integral membrane protein and an ATP-binding protein with homology to other transport proteins.     

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.     

Lantibiotics and microcins: polypeptides with unusual chemical diversity

Bacterial-derived antimicrobial polypeptides enjoy a large degree of structural and chemical diversity. Two well-studied examples of such polypeptides are the lanthionine-containing lantibiotics produced by a variety of Gram-positive bacteria, and their Gram-negative counterparts, the microcins. Both groups are produced as gene-encoded precursor peptides and undergo post-translational modification to generate the active moieties. Structure elucidation of novel lantibiotics and microcins has recently uncovered further novel structural and chemical features and, combined with the generation of analogue peptides by genetic manipulation, new insights into structure-function relationships have been gained. Furthermore, study of the mode of action of the lantibiotics nisin and mersacidin has revealed their use of a 'docking molecule' in the target cell to facilitate their biological activities. Meanwhile, in vitro studies with microcin B17 have helped to uncover the molecular mechanisms by which post-translational modification results in the formation of heterocyclic oxazole and thiazole rings. From a practical standpoint, both groups of polypeptides represent new lead structures for future development of antimicrobial agents, whilst the identification of the 'docking molecules' represents a step forward in the search for novel targets for future antibiosis.     

Plantazolicin, a novel microcin B17/streptolysin S-like natural product from Bacillus amyloliquefaciens FZB42

Here we report on a novel thiazole/oxazole-modified microcin (TOMM) from Bacillus amyloliquefaciens FZB42, a Gram-positive soil bacterium. This organism is well known for stimulating plant growth and biosynthesizing complex small molecules that suppress the growth of bacterial and fungal plant pathogens. Like microcin B17 and streptolysin S, the TOMM from B. amyloliquefaciens FZB42 undergoes extensive posttranslational modification to become a bioactive natural product. Our data show that the modified peptide bears a molecular mass of 1,335 Da and displays antibacterial activity toward closely related Gram-positive bacteria. A cluster of 12 genes that covers ～10 kb is essential for the production, modification, export, and self-immunity of this natural product. We have named this compound plantazolicin (PZN), based on the association of several producing organisms with plants and the incorporation of azole heterocycles, which derive from Cys, Ser, and Thr residues of the precursor peptide.     

The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.     

A Molecular Mechanics and Database Analysis of the Structural Preorganization and Activation of the Chromophore-Containing Hexapeptide Fragment in Green Fluorescent Protein

Abstract

We propose that heterologous posttranslational chromophore formation in green fluorescent protein (GFP) occurs because the chromophore-forming amino acid residues 65SYG67 are preorganized and activated for imidazolinone ring formation. Based on extensive molecular mechanical conformational searching of the precursor hexapeptide fragment (64FSYGVQ69), we suggest that the presence of low energy conformations characterized by short contacts (～3Å) between the carbonyl carbon of Ser65 and the amide nitrogen of Gly67 accounts for the initial step in posttranslational chromophore formation. Database searches showed that the tight turn required to establish the key short contact is a unique structural motif that is rarely found, except in other FSYG tetrapeptide sequences. Additionally, ab initio calculations demonstrated that an arginine side chain can hydrogen bond to the carbonyl oxygen of Ser65, activating this group for nucleophilic attack by the nearby lone pair of the Gly67 amide nitrogen. We propose that GFP chromophore-formation is initiated by a unique combination of conformational and electronic enhancements, identified by computational methods.     

The Ile13 residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA

Entry of the peptide antibiotic microcin J25 (MccJ25) into target cells is mediated by the outer membrane receptor FhuA and the inner membrane protein SbmA. The latter also transports MccB17 into the cell cytoplasm. Comparison of MccJ25 and MccB17 revealed a tetrapeptide sequence (VGIG) common to both antibiotics. We speculated that this structural feature in MccJ25 could be a motif recognized by SbmA. To test this hypothesis, we used a MccJ25 variant in which the isoleucine in VGIG (position 13 in the MccJ25 sequence) was replaced by lysine (I13K). In experiments in which the FhuA receptor was bypassed, the substituted microcin showed an inhibitory activity similar to that of the wild-type peptide. Moreover, MccJ25 interfered with colicin M uptake by FhuA in a competition assay, while the I13K mutant did not. From these results, we propose that the Ile¹³ residue is only required for interaction with FhuA, and that VGIG is not a major recognition element by SbmA.