Structural Basis of Cysteine Ligase MshC Inhibition by Cysteinyl-Sulfonamides.

Mycothiol (MSH), the major cellular thiol in Mycobacterium tuberculosis (Mtb), plays an essential role in the resistance of Mtb to various antibiotics and oxidative stresses. MshC catalyzes the ATP-dependent ligation of 1-O-(2-amino-2-deoxy-α-d-glucopyranosyl)-d-myo-inositol (GlcN-Ins) with l-cysteine (l-Cys) to form l-Cys-GlcN-Ins, the penultimate step in MSH biosynthesis. The inhibition of MshC is lethal to Mtb. In the present study, five new cysteinyl-sulfonamides were synthesized, and their binding affinity with MshC was evaluated using a thermal shift assay. Two of them bind the target with EC50 values of 219 and 231 µM. Crystal structures of full-length MshC in complex with these two compounds showed that they were bound in the catalytic site of MshC, inducing dramatic conformational changes of the catalytic site compared to the apo form. In particular, the observed closure of the KMSKS loop was not detected in the published cysteinyl-sulfamoyl adenosine-bound structure, the latter likely due to trypsin treatment. Despite the confirmed binding to MshC, the compounds did not suppress Mtb culture growth, which might be explained by the lack of adequate cellular uptake. Taken together, these novel cysteinyl-sulfonamide MshC inhibitors and newly reported full-length apo and ligand-bound MshC structures provide a promising starting point for the further development of novel anti-tubercular drugs targeting MshC.

Discovery of novel druggable pockets on polyomavirus VP1 through crystallographic fragment-based screening to develop capsid assembly inhibitors.

Polyomaviruses are a family of ubiquitous double-stranded DNA viruses many of which are human pathogens. These include BK polyomavirus which causes severe urinary tract infection in immunocompromised patients and Merkel cell polyomavirus associated with aggressive cancers. The small genome of polyomaviruses lacks conventional drug targets, and no specific drugs are available at present. Here we focus on the main structural protein VP1 of BK polyomavirus which is responsible for icosahedral capsid formation. To provide a foundation towards rational drug design, we crystallized truncated VP1 pentamers and subjected them to a high-throughput screening for binding drug-like fragments through a direct X-ray analysis. To enable a highly performant screening, rigorous optimization of the crystallographic pipeline and processing with the latest generation PanDDA2 software were necessary. As a result, a total of 144 binding hits were established. Importantly, the hits are well clustered in six surface pockets. Three pockets are located on the outside of the pentamer and map on the regions where the 'invading' C-terminal arm of another pentamer is attached upon capsid assembly. Another set of three pockets is situated within the wide pore along the five-fold axis of the VP1 pentamer. These pockets are situated at the interaction interface with the minor capsid protein VP2 which is indispensable for normal functioning of the virus. Here we systematically analyse the three outside pockets which are highly conserved across various polyomaviruses, while point mutations in these pockets are detrimental for viral replication. We show that one of the pockets can accommodate antipsychotic drug trifluoperazine. For each pocket, we derive pharmacophore features which enable the design of small molecules preventing the interaction between VP1 pentamers and therefore inhibiting capsid assembly. Our data lay a foundation towards a rational development of first-in-class drugs targeting polyomavirus capsid.

Partitioning of the initial catalytic steps of leucyl-tRNA synthetase is driven by an active site peptide-plane flip.

To correctly aminoacylate tRNALeu, leucyl-tRNA synthetase (LeuRS) catalyzes three reactions: activation of leucine by ATP to form leucyl-adenylate (Leu-AMP), transfer of this amino acid to tRNALeu and post-transfer editing of any mischarged product. Although LeuRS has been well characterized biochemically, detailed structural information is currently only available for the latter two stages of catalysis. We have solved crystal structures for all enzymatic states of Neisseria gonorrhoeae LeuRS during Leu-AMP formation. These show a cycle of dramatic conformational changes, involving multiple domains, and correlate with an energetically unfavorable peptide-plane flip observed in the active site of the pre-transition state structure. Biochemical analyses, combined with mutant structural studies, reveal that this backbone distortion acts as a trigger, temporally compartmentalizing the first two catalytic steps. These results unveil the remarkable effect of this small structural alteration on the global dynamics and activity of the enzyme.

Towards Novel 3-Aminopyrazinamide-Based Prolyl-tRNA Synthetase Inhibitors: In Silico Modelling, Thermal Shift Assay and Structural Studies.

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Phenyltriazole-functionalized sulfamate inhibitors targeting tyrosyl- or isoleucyl-tRNA synthetase.

Antimicrobial resistance is considered as one of the major threats for the near future as the lack of effective treatments for various infections would cause more deaths than cancer by 2050. The development of new antibacterial drugs is considered as one of the cornerstones to tackle this problem. Aminoacyl-tRNA synthetases (aaRSs) are regarded as good targets to establish new therapies. Apart from being essential for cell viability, they are clinically validated. Indeed, mupirocin, an isoleucyl-tRNA synthetase (IleRS) inhibitor, is already commercially available as a topical treatment for MRSA infections. Unfortunately, resistance developed soon after its introduction on the market, hampering its clinical use. Therefore, there is an urgent need for new cellular targets or improved therapies. Follow-up research by Cubist Pharmaceuticals led to a series of selective and in vivo active aminoacyl-sulfamoyl aryltetrazole inhibitors targeting IleRS (e.g. CB 168). Here, we describe the synthesis of new IleRS and TyrRS inhibitors based on the Cubist Pharmaceuticals compounds, whereby the central ribose was substituted for a tetrahydropyran ring. Various linkers were evaluated connecting the six-membered ring with the base-mimicking part of the synthesized analogues. Out of eight novel molecules, a three-atom spacer to the phenyltriazole moiety, which was established using azide-alkyne click chemistry, appeared to be the optimized linker to inhibit IleRS. However, 11 (Ki,app = 88 ± 5.3 nM) and 36a (Ki,app = 114 ± 13.5 nM) did not reach the same level of inhibitory activity as for the known high-affinity natural adenylate-intermediate analogue isoleucyl-sulfamoyl adenosine (IleSA, CB 138; Ki,app = 1.9 ± 4.0 nM) and CB 168, which exhibit a comparable inhibitory activity as the native ligand. Therefore, 11 was docked into the active site of IleRS using a known crystal structure of T. thermophilus in complex with mupirocin. Here, we observed the loss of the crucial 3'- and 4'- hydroxyl group interactions with the target enzyme compared to CB 168 and mupirocin, which we suggest to be the reason for the limited decrease in enzyme affinity. Despite the lack of antibacterial activity, we believe that structurally optimizing these novel analogues via a structure-based approach could ultimately result in aaRS inhibitors which would help to tackle the antibiotic resistance problem.

Synthesis and Biological Evaluation of Lipophilic Nucleoside Analogues as Inhibitors of Aminoacyl-tRNA Synthetases.

Emerging antibiotic resistance in pathogenic bacteria and reduction of compounds in the existing antibiotics discovery pipeline is the most critical concern for healthcare professionals. A potential solution aims to explore new or existing targets/compounds. Inhibition of bacterial aminoacyl-tRNA synthetase (aaRSs) could be one such target for the development of antibiotics. The aaRSs are a group of enzymes that catalyze the transfer of an amino acid to their cognate tRNA and therefore play a pivotal role in translation. Thus, selective inhibition of these enzymes could be detrimental to microbes. The 5'-O-(N-(L-aminoacyl)) sulfamoyladenosines (aaSAs) are potent inhibitors of the respective aaRSs, however due to their polarity and charged nature they cannot cross the bacterial membranes. In this work, we increased the lipophilicity of these existing aaSAs in an effort to promote their penetration through the bacterial membrane. Two strategies were followed, either attaching a (permanent) alkyl moiety at the adenine ring via alkylation of the N6-position or introducing a lipophilic biodegradable prodrug moiety at the alpha-terminal amine, totaling eight new aaSA analogues. All synthesized compounds were evaluated in vitro using either a purified Escherichia coli aaRS enzyme or in presence of total cellular extract obtained from E. coli. The prodrugs showed comparable inhibitory activity to the parent aaSA analogues, indicating metabolic activation in cellular extracts, but had little effect on bacteria. During evaluation of the N6-alkylated compounds against different microbes, the N6-octyl containing congener 6b showed minimum inhibitory concentration (MIC) of 12.5 µM against Sarcina lutea while the dodecyl analogue 6c displayed MIC of 6.25 µM against Candida albicans.

Acylated sulfonamide adenosines as potent inhibitors of the adenylate-forming enzyme superfamily.

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.

Comparative analysis of pyrimidine substituted aminoacyl-sulfamoyl nucleosides as potential inhibitors targeting class I aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) catalyse the ATP-dependent coupling of an amino acid to its cognate tRNA. Being vital for protein translation aaRSs are considered a promising target for the development of novel antimicrobial agents. 5'-O-(N-aminoacyl)-sulfamoyl adenosine (aaSA) is a non-hydrolysable analogue of the aaRS reaction intermediate that has been shown to be a potent inhibitor of this enzyme family but is prone to chemical instability and enzymatic modification. In an attempt to improve the molecular properties of this scaffold we synthesized a series of base substituted aaSA analogues comprising cytosine, uracil and N3-methyluracil targeting leucyl-, tyrosyl- and isoleucyl-tRNA synthetases. In in vitro assays seven out of the nine inhibitors demonstrated Kiapp values in the low nanomolar range. To complement the biochemical studies, X-ray crystallographic structures of Neisseria gonorrhoeae leucyl-tRNA synthetase and Escherichia coli tyrosyl-tRNA synthetase in complex with the newly synthesized compounds were determined. These highlighted a subtle interplay between the base moiety and the target enzyme in defining relative inhibitory activity. Encouraged by this data we investigated if the pyrimidine congeners could escape a natural resistance mechanism, involving acetylation of the amine of the aminoacyl group by the bacterial N-acetyltransferases RimL and YhhY. With RimL the pyrimidine congeners were less susceptible to inactivation compared to the equivalent aaSA, whereas with YhhY the converse was true. Combined the various insights resulting from this study will pave the way for the further rational design of aaRS inhibitors.

Family-wide analysis of aminoacyl-sulfamoyl-3-deazaadenosine analogues as inhibitors of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are enzymes that precisely attach an amino acid to its cognate tRNA. This process, which is essential for protein translation, is considered a viable target for the development of novel antimicrobial agents, provided species selective inhibitors can be identified. Aminoacyl-sulfamoyl adenosines (aaSAs) are potent orthologue specific aaRS inhibitors that demonstrate nanomolar affinities in vitro but have limited uptake. Following up on our previous work on substitution of the base moiety, we evaluated the effect of the N3-position of the adenine by synthesizing the corresponding 3-deazaadenosine analogues (aaS3DAs). A typical organism has 20 different aaRS, which can be split into two distinct structural classes. We therefore coupled six different amino acids, equally targeting the two enzyme classes, via the sulfamate bridge to 3-deazaadenosine. Upon evaluation of the inhibitory potency of the obtained analogues, a clear class bias was noticed, with loss of activity for the aaS3DA analogues targeting class II enzymes when compared to the equivalent aaSA. Evaluation of the available crystallographic structures point to the presence of a conserved water molecule which could have importance for base recognition within class II enzymes, a property that can be explored in future drug design efforts.

5'-(N-aminoacyl)-sulfonamido-5'-deoxyadenosine: attempts for a stable alternative for aminoacyl-sulfamoyl adenosines as aaRS inhibitors.

Synthesis of aminoacyl-sulfamoyl adenosines (aaSAs) and their peptidyl conjugates as aminoacyl tRNA synthetase (aaRS) inhibitors remains problematic due to the low yield of the aminoacylation and the subsequent conjugation reaction causing concomitant formation of a cyclic adenosine derivative. In an effort to reduce this undesirable side reaction, we aimed to prepare the corresponding aminoacyl sulfonamide (aaSoA) analogues as more stable alternatives for aaSA derivatives. Deletion of the 5'-oxygen in aaSA analogues should render the C-5' less electrophilic and therefore improve the stability of the aminoacyl sulfamate analogues. We therefore synthesized six sulfonamides and compared their activity against the respective aaSA analogues. However, except for the aspartyl derivative, the new compounds are not able to inhibit the corresponding aaRS. Possible reasons for this loss of activity are discussed by modeling and comparison of the newly synthesized aaSoA derivatives with their parent aaSA analogues.

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.

Base substituted 5'-O-(N-isoleucyl)sulfamoyl nucleoside analogues as potential antibacterial agents.

Aminoacyl-sulfamoyl adenosines are well-known nanomolar inhibitors of the corresponding prokaryotic and eukaryotic tRNA synthetases in vitro. Inspired by the aryl-tetrazole containing compounds of Cubist Pharmaceuticals and the modified base as found in the natural antibiotic albomycin, the selectivity issue of the sulfamoylated adenosines prompted us to investigate the pharmacophoric importance of the adenine base. We therefore synthesized and evaluated several isoleucyl-sulfamoyl nucleoside analogues with either uracil, cytosine, hypoxanthine, guanine, 1,3-dideaza-adenine (benzimidazole) or 4-nitro-benzimidazole as the heterocyclic base. Based on the structure and antibacterial activity of microcin C, we also prepared their hexapeptidyl conjugates in an effort to improve their uptake potential. We further compared their antibacterial activity with the parent isoleucyl-sulfamoyl adenosine (Ile-SA), both in in vitro and in cellular assays. Surprisingly, the strongest in vitro inhibition was found for the uracil containing analogue 16f. Unfortunately, only very weak growth inhibitory properties were found as of low uptake. The results are discussed in the light of previous literature findings.

In search of Flavivirus inhibitors part 2: tritylated, diphenylmethylated and other alkylated nucleoside analogues.

Several flaviviruses, such as the yellow fever virus and the dengue virus cause severe and potentially lethal infection in man. Following up on our initial hit 3',5'-bistritylated uridine 1, a series of alkylated nucleoside analogues were synthesized and evaluated for their in vitro antiviral activities against dengue fever virus and yellow fever virus. Hereto, alkyl and aryl groups were attached at various positions of the sugar ring combined with subtle variation of the heterocyclic base. Among the new series of derivatives, 3',5'-di-O-trityl-5-fluoro-2'-deoxyuridine (39) was the most efficient in this series and inhibited both yellow fever virus and dengue virus replication with a 50% effective concentration (EC50) of ∼1 µg/mL without considerable cytotoxicity. The other fluorinated derivatives proved more toxic. Almost all diphenylmethylated pyrimidine nucleosides with 3',5'-di-O-benzhydryl-2'-deoxyuridine (50) as the example were endowed with strong cytotoxic effects down to 1 µg/mL.

N-alkylated aminoacyl sulfamoyladenosines as potential inhibitors of aminoacylation reactions and microcin C analogues containing D-amino acids.

Microcin C analogues were recently envisaged as important compounds for the development of novel antibiotics. Two issues that may pose problems to these potential antibiotics are possible acquisition of resistance through acetylation and in vivo instability of the peptide chain. N-methylated aminoacyl sulfamoyladenosines were synthesized to investigate their potential as aminoacyl tRNA synthetase inhibitors and to establish whether these N-alkylated analogues would escape the natural inactivation mechanism via acetylation of the alpha amine. It was shown however, that these compounds are not able to effectively inhibit their respective aminoacyl tRNA synthetase. In addition, we showed that (D)-aspartyl-sulfamoyladenosine (i.e. with a (D)-configuration for the aspartyl moiety), is a potent inhibitor of aspartyl tRNA synthetase. However, we also showed that the inhibitory effect of (D)- aspartyl-sulfamoyladenosine is relatively short-lasting. Microcin C analogues with (D)-amino acids throughout from positions two to six proved inactive. They were shown to be resistant against metabolism by the different peptidases and therefore not able to release the active moiety. This observation could not be reversed by incorporation of (L)-amino acids at position six, showing that none of the available peptidases exhibit endopeptidase activity.

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.

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.

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.

The mechanism of microcin C resistance provided by the MccF peptidase.

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. Inside a sensitive cell, McC is processed by aminopeptidases, which release a nonhydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. The mccABCDE operon is sufficient for McC production and resistance of the producing cell to McC. An additional gene, mccF, which is adjacent to but not part of the mccABCDE operon, also provides resistance to exogenous McC. MccF is similar to Escherichia coli LdcA, an L,D-carboxypeptidase whose substrate is monomeric murotetrapeptide L-Ala-D-Glu-meso-A(2)pm-D-Ala or its UDP-activated murein precursor. The mechanism by which MccF provides McC resistance remained unknown. Here, we show that MccF detoxifies both intact and processed McC by cleaving an amide bond between the C-terminal aspartate and the nucleotide moiety. MccF also cleaves the same bond in nonhydrolyzable aminoacyl sulfamoyl adenosines containing aspartyl, glutamyl, and, to a lesser extent, seryl aminoacyl moieties but is ineffective against other aminoacyl adenylates.

MccE provides resistance to protein synthesis inhibitor microcin C by acetylating the processed form of the antibiotic.

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. McC is excreted from producing cells by the MccC transporter. The residual McC that remains in the producing cell can be processed by cellular aminopeptidases with the release of a non-hydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. Accumulation of processed McC inside producing cells should therefore lead to translation inhibition and cessation of growth. Here, we show that a product of another gene of the McC biosynthetic cluster, mccE, acetylates processed McC and converts it into a non-toxic compound. MccE also makes Escherichia coli resistant to albomycin, a Trojan horse inhibitor unrelated to McC that, upon processing, gives rise to a serine coupled to a thioxylofuranosyl pyrimidine, an inhibitor of seryl-tRNA synthetase. We speculate that MccE and related cellular acetyltransferases of the Rim family may detoxify various aminoacyl-nucleotides, either exogenous or those generated inside the cell.