Microcin C: biosynthesis, mode of action, and potential as a lead in antibiotics development

The natural compound Microcin C (McC) is a Trojan horse inhibitor of aspartyl tRNA synthetases endowed with strong antibacterial properties, in which a heptapeptide moiety is responsible for active transport of the inhibitory metabolite part into the bacterial cell. The intracellularly formed aspartyl AMP analogue carries a chemically more stable phosphoramidate linkage, in comparison to the labile aspartyl-adenylate, and in addition is esterified with a 3-aminopropyl moiety. Therefore, this compound can target aspartyl-tRNA synthetase. The biochemical production and secretion of McC, and the possibilities to develop new classes of antibiotics using the McC Trojan horse concept in combination with sulfamoylated adenosine analogues will be discussed briefly.     

Amino acid residues required for maturation, cell uptake, and processing of translation inhibitor microcin C

Microcin C (McC), a peptide-nucleotide Trojan horse antibiotic, targets aspartyl-tRNA synthetase. We present the results of a systematic mutational study of the 7-amino-acid ribosomally synthesized peptide moiety of McC. Our results define amino acid positions important for McC maturation and cell uptake and processing and open the way for creation of more potent McC-based inhibitors.     

Peptide‐nucleotide antibiotic Microcin C is a potent inducer of stringent response and persistence in both sensitive and producing cells

Microcin C (McC) is a peptide‐nucleotide antibiotic that inhibits aspartyl‐tRNA synthetase. Here, we show that McC is a strong inducer of persistence in Escherichia coli. Persistence induced by McC is mediated by (p)ppGpp and requires chromosomally encoded toxin‐antitoxin modules. McC‐producing cells have increased persistence levels due to a combined effect of McC imported from the cultured medium and intracellularly synthesized antibiotic. McC‐producing cells also induce persistence in sensitive cells during co‐cultivation, underscoring complex interactions in bacterial communities where an antagonistic compound produced by one community member can benefit other members by increasing their ability to withstand antibiotics.     

Synthetic Microcin C Analogs Targeting Different Aminoacyl-tRNA Synthetases

Microcin C (McC) is a potent antibacterial agent produced by some strains of Escherichia coli. McC consists of a ribosomally synthesized heptapeptide with a modified AMP attached through a phosphoramidate linkage to the α-carboxyl group of the terminal aspartate. McC is a Trojan horse inhibitor: it is actively taken inside sensitive cells and processed there, and the product of processing, a nonhydrolyzable aspartyl-adenylate, inhibits translation by preventing aminoacylation of tRNA^Asp by aspartyl-tRNA synthetase (AspRS). Changing the last residue of the McC peptide should result in antibacterial compounds with targets other than AspRS. However, mutations that introduce amino acid substitutions in the last position of the McC peptide abolish McC production. Here, we report total chemical synthesis of three McC-like compounds containing a terminal aspartate, glutamate, or leucine attached to adenosine through a nonhydrolyzable sulfamoyl bond. We show that all three compounds function in a manner similar to that of McC, but the first compound inhibits bacterial growth by targeting AspRS while the latter two inhibit, respectively, GluRS and LeuRS. Our approach opens a way for creation of new antibacterial Trojan horse agents that target any 1 of the 20 tRNA synthetases in the cell.Microcins are small (<10-kDa) ribosomally synthesized peptide antibiotics produced by Enterobacteriaceae (17). Three microcins, B, C, and J, form a subgroup of posttranslationally modified microcins. Members of this subgroup have highly unusual structures and inhibit cellular enzymes that are validated targets for antibacterial drug development (25). Posttranslationally modified microcins are attractive as drug candidates because of their strong antibacterial action and because virtually limitless numbers of their derivatives can be generated by means of mutation, chemical synthesis, or both. Microcin B (McB), a 43-residue peptide with thiazole and indole rings (13), inhibits DNA gyrase (21). Microcin J, a 21-amino-acid peptide, assumes an unusual threaded lasso structure (2, 23, 27) and inhibits bacterial RNA polymerase (1, 18). The structure of the subject of this study, McC (compound 1) is shown in Fig. ​Fig.1a.1a. McC is a heptapeptide with a formylated N-terminal methionine and a C-terminal aspartate whose α-carboxyl group is covalently linked to adenosine through an N-acyl phosphoramide bond (10, 14). The phosphoramidate of McC is additionally modified by an O-propylamine group (9).Open in a separate windowFIG. 1.Structures and synthesis of McC analogs. (a) Structures of microcin C (compound 1) and its processing product (compound 2). (b) Structures of synthetic McC analogs 7 to 9 and their expected processing products, compounds 4 to 6, which are established inhibitors of AspRS, GluRS, and LeuRS, respectively. (c) Structure of Asp-AMP (compound 3), the natural reaction intermediate of AspRS. Compounds 2 and 4 are nonhydrolyzable analogs of this compound. (d) Synthesis of compounds 7 to 9, which starts from compounds 4 to 6. Hereto the hexapeptide was coupled to the sulfamoyl precursors 4-6 via the coupling agent DIC, followed by removal of the Fmoc protecting group: (i) Fmoc-MRTGNA-OH, HOBt, DIC, DIPEA; (ii) Et₃N/DMF (1:1 [vol/vol]).The passage of McC through the inner layer of the Escherichia coli cell wall is carried out by the YejABEF transporter (19). Once inside the cell, McC is specifically processed by one of the several broad-specificity E. coli cytoplasmic aminopeptidases (12). The product of processing, modified aspartyl-adenylate (compound 2) (15), closely resembles Asp-AMP (compound 3) (Fig. ​(Fig.1c),1c), the natural reaction intermediate of the tRNA^Asp aminoacylation reaction catalyzed by AspRS. However, because the bond between the α-carboxyl of C-terminal aspartate and the phosphoramidate nitrogen is nonhydrolyzable, compound 2 inhibits AspRS. Unprocessed McC has no effect on tRNA^Asp aminoacylation, while processed McC has no effect on McC-sensitive cells at concentrations at which intact McC strongly inhibits cell growth. Thus, McC is a Trojan horse inhibitor (22): the peptide part allows McC to enter sensitive cells, where it gets processed, liberating the inhibitory part of the drug.Aminoacyl-tRNA synthetases (aaRSs) carry out the condensation of genetically encoded amino acids with cognate tRNAs. When 1 of the 20 aaRSs present in the cell is inhibited, the corresponding tRNA is not charged. This leads to protein synthesis inhibition and cell growth arrest. In principle, variation of the last amino acid of the McC peptide, the product of the mccA gene, should allow investigators to obtain McC derivatives targeting aaRSs other than AspRS. Unfortunately, the results of systematic structure-activity analyses of the McC peptide revealed that substitutions in the seventh codon of mccA invariably prevented McC production, presumably by interfering with posttranslational modifications of the MccA peptide by the McC maturation enzymes (11). Indeed, in vitro analysis showed that the C-terminal asparagine of MccA is required for the addition of the adenosine moiety by the MccB protein (24).Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of their corresponding aaRSs (5, 20, 26). However, these compounds show low in vivo activities due to limited membrane permeability and the absence of a transporter for these compounds. Here, we show that through chemical attachment of aminoacyl-sulfamoyl adenosines to the first 6 amino acids of the MccA peptide, potent antibacterial agents can be generated. The new compounds share the Trojan horse mechanism of action with McC but target aaRSs specified by the last amino acid of the peptide moiety.     

Characterization of peptide chain length and constituency requirements for YejABEF-mediated uptake of microcin C analogues

Microcin C (McC), a natural antibacterial compound consisting of a heptapeptide attached to a modified adenosine, is actively taken up by the YejABEF transporter, after which it is processed by cellular aminopeptidases, releasing the nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC analogues with variable length of the peptide moiety were synthesized and evaluated in order to characterize the substrate preferences of the YejABEF transporter. It was shown that a minimal peptide chain length of 6 amino acids and the presence of an N-terminal formyl-methionyl-arginyl sequence are required for transport.     

Microcin C: biosynthesis and mechanisms of bacterial resistance

Nonhydrolyzable aminoacyl-adenylates that inhibit protein synthesis provide a promising route towards the development of novel antibiotics whose mechanism of action limits the appearance of bacterial drug resistance. The 'Trojan horse' antibiotic microcin C (McC) consists of a nonhydrolyzable aspartyl-adenylate that is efficiently imported into bacterial cells owing to a covalently attached peptide carrier. Once inside the cell, the carrier is removed by proteolytic processing to release a potent aspartyl tRNA synthetase inhibitor. The focus of this article is on the mechanism of biosynthesis of McC. We also examine the strategies utilized by McC-producing strains to overcome toxicity due to unwanted, premature processing of the drug. This article will discuss how McC biosynthesis can be systematically manipulated for the development of derivatives that will target the entire battery of aminoacyl tRNA synthetases in various bacteria.     

Structural and functional characterization of microcin C resistance peptidase MccF from Bacillus anthracis

Microcin C (McC) is heptapeptide adenylate antibiotic produced by Escherichia coli strains carrying the mccABCDEF gene cluster encoding enzymes, in addition to the heptapeptide structural gene mccA, necessary for McC biosynthesis and self-immunity of the producing cell. The heptapeptide facilitates McC transport into susceptible cells, where it is processed releasing a non-hydrolyzable aminoacyl adenylate that inhibits an essential aminoacyl-tRNA synthetase. The self-immunity gene mccF encodes a specialized serine peptidase that cleaves an amide bond connecting the peptidyl or aminoacyl moieties of, respectively, intact and processed McC with the nucleotidyl moiety. Most mccF orthologs from organisms other than E. coli are not linked to the McC biosynthesis gene cluster. Here, we show that a protein product of one such gene, MccF from Bacillus anthracis (BaMccF), is able to cleave intact and processed McC, and we present a series of structures of this protein. Structural analysis of apo-BaMccF and its adenosine monophosphate complex reveals specific features of MccF-like peptidases that allow them to interact with substrates containing nucleotidyl moieties. Sequence analyses and phylogenetic reconstructions suggest that several distinct subfamilies form the MccF clade of the large S66 family of bacterial serine peptidases. We show that various representatives of the MccF clade can specifically detoxify non-hydrolyzable aminoacyl adenylates differing in their aminoacyl moieties. We hypothesize that bacterial mccF genes serve as a source of bacterial antibiotic resistance.     

Extended targeting potential and improved synthesis of Microcin C analogs as antibacterials

Microcin C (McC) (1) is a potent antibacterial compound produced by some Escherichia coli strains. McC functions through a Trojan-Horse mechanism: it is actively taken up inside a sensitive cell through the function of the YejABEF-transporter and then processed by cellular aminopeptidases. Processed McC (2) is a non-hydrolysable aspartyl-adenylate analog that inhibits aspartyl-tRNA synthetase (AspRS). A new synthesis is described that allows for the production of a wide variety of McC analogs in acceptable amounts. Using this synthesis a number of diverse compounds was synthesized with altered target specificity. Further characteristics of the YejABEF transporters were determined using these compounds.     

Escherichia coli peptidase A, B, or N can process translation inhibitor microcin C

The heptapeptide-nucleotide microcin C (McC) targets aspartyl-tRNA synthetase. Upon its entry into a susceptible cell, McC is processed to release a nonhydrolyzable aspartyl-adenylate that inhibits aspartyl-tRNA synthetase, leading to the cessation of translation and cell growth. Here, we surveyed Escherichia coli cells with singly, doubly, and triply disrupted broad-specificity peptidase genes to show that any of three nonspecific oligopeptidases (PepA, PepB, or PepN) can effectively process McC. We also show that the rate-limiting step of McC processing in vitro is deformylation of the first methionine residue of McC.     

Structural basis for microcin C7 inactivation by the MccE acetyltransferase

The antibiotic microcin C7 (McC) acts as a bacteriocide by inhibiting aspartyl-tRNA synthetase and stalling the protein translation machinery. McC is synthesized as a heptapeptide-nucleotide conjugate, which is processed by cellular peptidases within target strains to yield the biologically active compound. As unwanted processing of intact McC can result in self-toxicity, producing strains utilize multiple mechanisms for autoimmunity against processed McC. We have shown previously that the mccE gene within the biosynthetic cluster can inactivate processed McC by acetylating the antibiotic. Here, we present the characterization of this acetylation mechanism through biochemical and structural biological studies of the MccE acetyltransferase domain (MccE(AcTase)). We have also determined five crystal structures of the MccE-acetyl-CoA complex with bound substrates, inhibitor, and reaction product. The structural data reveal an unexpected mode of substrate recognition through π-stacking interactions similar to those found in cap-binding proteins and nucleotidyltransferases. These studies provide a rationale for the observation that MccE(AcTase) can detoxify a range of aminoacylnucleotides, including those that are structurally distinct from microcin C7.     

Structural characterization of tatiopterin, a novel pterin isolated from Methanogenium tationis

Cofactor extracts of Methanogenium tationis were screened for the presence of pterin-derivatives. Methanopterin, sarcinapterin and 7-methylpterin were absent, while 2-amino-4-hydroxy-pteridine and another blue fluorescent compound with a pterin spectrum were detected. The latter pterin was purified by ion exchange and reversed-phase column chromatography. The structure of this compound was elucidated by combining spectrophotometry, amino acid analysis and 1H-NMR spectroscopy. The pterin, which we named tatiopterin, was identified as an aspartyl derivative of sarcinapterin with a 7-proton instead of a 7-methyl group in the pterin moiety. The IUPAC name is: N-[-1'-(2'-amino-4'-hydroxy-7'-proton-6'-pteridinyl)ethyl]-4- [2',3',4',5'-tetrahydroxypent-1'-yl(5'----1')O-alpha- ribofuranosyl-5'-phosphoric acid]aniline, in which the phosphate group is esterified with alpha-hydroxyglutarylglutamylaspartic acid.     

The RimL Transacetylase Provides Resistance to Translation Inhibitor Microcin C

Peptide-nucleotide antibiotic microcin C (McC) is produced by some Escherichia coli strains. Inside a sensitive cell, McC is processed, releasing a nonhydrolyzable analog of aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. The product of mccE, a gene from the plasmid-borne McC biosynthetic cluster, acetylates processed McC, converting it into a nontoxic compound. MccE is homologous to chromosomally encoded acetyltransferases RimI, RimJ, and RimL, which acetylate, correspondingly, the N termini of ribosomal proteins S18, S5, and L12. Here, we show that E. coli RimL, but not other Rim acetyltransferases, provides a basal level of resistance to McC and various toxic nonhydrolyzable aminoacyl adenylates. RimL acts by acetylating processed McC, which along with ribosomal protein L12 should be considered a natural RimL substrate. When overproduced, RimL also makes cells resistant to albomycin, an antibiotic that upon intracellular processing gives rise to a seryl-thioribosyl pyrimidine that targets seryl-tRNA synthetase. We further show that E. coli YhhY, a protein related to Rim acetyltransferases but without a known function, is also able to detoxify several nonhydrolyzable aminoacyl adenylates but not processed McC. We propose that RimL and YhhY protect bacteria from various toxic aminoacyl nucleotides, either exogenous or those generated inside the cell during normal metabolism.     

P-O bond destabilization accelerates phosphoenzyme hydrolysis of sarcoplasmic reticulum Ca2+ -ATPase

The phosphate group of the ADP-insensitive phosphoenzyme (E2-P) of sarcoplasmic reticulum Ca2+ -ATPase (SERCA1a) was studied with infrared spectroscopy to understand the high hydrolysis rate of E2-P. By monitoring an autocatalyzed isotope exchange reaction, three stretching vibrations of the transiently bound phosphate group were selectively observed against a background of 50,000 protein vibrations. They were found at 1194, 1137, and 1115 cm(-1). This information was evaluated using the bond valence model and empirical correlations. Compared with the model compound acetyl phosphate, structure and charge distribution of the E2-P aspartyl phosphate resemble somewhat the transition state in a dissociative phosphate transfer reaction; the aspartyl phosphate of E2-P has 0.02 A shorter terminal P-O bonds and a 0.09 A longer bridging P-O bond that is approximately 20% weaker, the angle between the terminal P-O bonds is wider, and -0.2 formal charges are shifted from the phosphate group to the aspartyl moiety. The weaker bridging P-O bond of E2-P accounts for a 10(11)-10(15)-fold hydrolysis rate enhancement, implying that P-O bond destabilization facilitates phosphoenzyme hydrolysis. P-O bond destabilization is caused by a shift of noncovalent interactions from the phosphate oxygens to the aspartyl oxygens. We suggest that the relative positioning of Mg2+ and Lys684 between phosphate and aspartyl oxygens controls the hydrolysis rate of the ATPase phosphoenzymes and related phosphoproteins.     

Identification of human erythrocyte blood group antigens on the C3b/C4b receptor.

The Knops/McCoy (Kn/McC) human erythrocyte blood group system belongs to the category of blood group Ag that generate so-called "high titer low avidity" antibodies in immunized transfusion recipients. Screening of red cells lacking certain high titer low avidity Ag demonstrated markedly diminished CR1 expression on McC(d-) and Kn/McC "null" (Kn(a-)McC(a-b-c-d-e-f-] erythrocytes. Additional testing by other methods confirmed these data, and biochemical assays demonstrated no detectable immunoreactive CR1 protein in membranes from Kn/McC null red cells. Human antisera to various Kn/McC Ag were then used to demonstrate that many of these antisera could be used to isolate a protein of identical m.w. to that isolated from the same cells using murine mAb CR1 antisera. Finally, protein isolated by using murine mAb anti-CR1 reacted specifically with anti-Kn/McC antibodies, demonstrating the identity of the Kn/McC and CR1 proteins. Thus, CR1 protein bears the human erythrocyte Kn/McC blood group Ag.     

The achilles heel of the trojan horse model of HIV-1 trans-infection

To ensure their survival, microbial pathogens have evolved diverse strategies to subvert host immune defenses. The human retrovirus HIV-1 has been proposed to hijack the natural endocytic function of dendritic cells (DCs) to infect interacting CD4 T cells in a process termed trans-infection. Although DCs can be directly infected by certain strains of HIV-1, productive infection of DCs is not required during trans-infection; instead, DCs capture and internalize infectious HIV-1 virions in vesicles for later transmission to CD4 T cells via vesicular exocytosis across the infectious synapse. This model of sequential endocytosis and exocytosis of intact HIV-1 virions has been dubbed the "Trojan horse" model of HIV-1 trans-infection. While this model gained rapid favor as a strong example of how a pathogen exploits the natural properties of its cellular host, our recent studies challenge this model by showing that the vast majority of virions transmitted in trans originate from the plasma membrane rather than from intracellular vesicles. This review traces the experimental lines of evidence that have contributed to what we view as the "rise and decline" of the Trojan horse model of HIV-1 trans-infection.     

Identification of a novel tatiopterin derivative in Methanogenium tationis

Recently, a novel pterin has been isolated from Methanogenium tationis. This pterin derivative, which was called tatiopterin, was characterized as a methanopterin-like structure with an additional aspartyl and glutamyl group in the side chain and with a 7-proton instead of a 7-methyl group in the pterin moiety. The sequence of the aspartyl and glutamyl group remained unsolved. In this study, a novel pterin was purified from Mg.tationis and analyzed by 600 MHz 1H-NMR spectroscopy and fast atom bombardment-mass spectroscopy. This pterin was found to be an aspartyl derivative of methanopterin with a 7-proton in the pterin part of the molecule. No glutamyl group could be detected. Apparently, Mg.tationis is able to synthesize two types of tatiopterin derivatives. For these cofactors the trivial names 'tatiopterin-0' (lacking a glutamyl group) and 'tatiopterin-I' (containing one glutamyl group) are introduced here.     

Structure guided P1' modifications of HEA derived β-secretase inhibitors for the treatment of Alzheimer's disease

The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with potential off target toxicity, and improved microsomal stability is exemplified.     

The Escherichia coli Yej transporter is required for the uptake of translation inhibitor microcin C

Microcin C (McC), a peptide-nucleotide antibiotic, targets aspartyl-tRNA synthetase. By analyzing a random transposon library, we identified Escherichia coli mutants resistant to McC. Transposon insertions were localized to a single locus, yejABEF, which encodes components of a putative inner membrane ABC transporter. Analysis of site-specific mutants established that all four components of the transporter are required for McC sensitivity. Since aspartyl-tRNA synthetase in yej mutant extracts was fully sensitive to McC, we conclude that yej mutations interfere with McC uptake and that YejABEF is the only inner membrane transporter responsible for McC uptake in E. coli. Other substrates of YejABEF remain to be identified.     

Neocarzinostatin-based hybrid biocatalysts with a RNase like activity

A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N′(17′-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called ‘Trojan horse’ strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (k_cat/K_M = 13.6 M⁻¹ s⁻¹ at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the ‘Trojan horse’ strategy for obtaining better catalysts of selective reactions will be further studied.