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The FEBS Journal Jul 2009The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)-dependent and alpha-ketoglutarate-dependent Cbeta-hydroxylation of L-arginine during the...
The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)-dependent and alpha-ketoglutarate-dependent Cbeta-hydroxylation of L-arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substrate L-arginine, the product (2S,3S)-hydroxyarginine and the coproduct succinate at 1.1-1.3 A resolution. The overall structure reveals a beta-helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase-like superfamily. In contrast to other clavaminic acid synthase-like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cbeta-hydroxylated L-arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(-) conformer for chi(1) instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase-like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of the L-arginine substrate projects outwards from the active site by undergoing interactions mainly with the C-terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogs L-homoarginine and L-canavanine for Cbeta-hydroxylation.
Topics: Anti-Bacterial Agents; Arginine; Bacterial Proteins; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Molecular Sequence Data; Molecular Structure; Nonheme Iron Proteins; Oxygenases; Protein Structure, Secondary; Protein Structure, Tertiary; Stereoisomerism; Streptomyces; Substrate Specificity; Viomycin
PubMed: 19490124
DOI: 10.1111/j.1742-4658.2009.07085.x -
Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans.Chembiochem : a European Journal of... Jan 2009Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is...
Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin.
Topics: Amino Acids; Antitubercular Agents; Bacterial Proteins; Gene Deletion; Multigene Family; Streptomyces lividans; Viomycin
PubMed: 19105177
DOI: 10.1002/cbic.200800646 -
Molecular Microbiology Jul 2008The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation....
The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.
Topics: Colicins; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Models, Biological; Protein Biosynthesis; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Transfer; Ribosomes
PubMed: 18485067
DOI: 10.1111/j.1365-2958.2008.06283.x -
Biophysical Journal Nov 2007Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states...
Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg(2+)], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.
Topics: Biophysics; Fluorescence Resonance Energy Transfer; Ions; Magnesium; Models, Chemical; Nucleic Acid Conformation; Peptide Chain Elongation, Translational; Peptides; Polyethylene Glycols; Protein Transport; RNA, Transfer; RNA, Transfer, Amino Acyl; Ribosomes; Time Factors; Viomycin
PubMed: 17693476
DOI: 10.1529/biophysj.107.109884 -
Molecular Cell Feb 2007Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one...
Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.
Topics: Anti-Bacterial Agents; Escherichia coli; Fluorescence; Guanosine Triphosphate; Hydrolysis; Kinetics; Models, Genetic; Mutation; N-Formylmethionine; Nucleic Acid Conformation; Peptide Elongation Factor G; Protein Biosynthesis; Puromycin; RNA Transport; RNA, Transfer
PubMed: 17317625
DOI: 10.1016/j.molcel.2007.01.014 -
Journal of Natural Products Apr 2007The nonproteinogenic amino acid capreomycidine is the signature residue found in the tuberactinomycin family of antitubercular peptide antibiotics and an important...
The nonproteinogenic amino acid capreomycidine is the signature residue found in the tuberactinomycin family of antitubercular peptide antibiotics and an important element of the pharmacophore. Recombinant VioG, a single-module peptide synthetase from the viomycin gene cluster cloned from Streptomyces vinaceus (ATCC11861), specifically activates capreomycidine for incorporation into viomycin (tuberactinomycin B). Insertional disruption of the putative hydroxylase gene vioQ resulted in a mutant that accumulated tuberactinomycin O, suggesting that hydroxylation at C-5 of the capreomycidine residue is a post-assembly event. The inactivated chromosomal copy of vioQ could be complemented with a wild-type copy of the gene to restore viomycin production.
Topics: Anti-Bacterial Agents; Base Sequence; Escherichia coli; Genes, Bacterial; Molecular Structure; Peptide Synthases; Streptomyces; Viomycin
PubMed: 17302456
DOI: 10.1021/np060605u -
Molecular Cell Jul 2006The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent...
The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.
Topics: Antibiotics, Antitubercular; Bacterial Proteins; Base Sequence; Capreomycin; Cloning, Molecular; Drug Resistance, Bacterial; Escherichia coli; Mass Spectrometry; Methylation; Molecular Sequence Data; Mutation; Mycobacteriaceae; Nucleic Acid Conformation; RNA, Bacterial; RNA, Ribosomal, 16S; RNA, Ribosomal, 28S; Recombinant Proteins; Ribosomes
PubMed: 16857584
DOI: 10.1016/j.molcel.2006.05.044 -
Antimicrobial Agents and Chemotherapy Aug 2005Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them...
Capreomycin, kanamycin, amikacin, and viomycin are drugs that are used to treat multidrug-resistant tuberculosis. Each inhibits translation, and cross-resistance to them is a concern during therapy. A recent study revealed that mutation of the tlyA gene, encoding a putative rRNA methyltransferase, confers capreomycin and viomycin resistance in Mycobacterium tuberculosis bacteria. Mutations in the 16S rRNA gene (rrs) have been associated with resistance to each of the drugs; however, reports of cross-resistance to the drugs have been variable. We investigated the role of rrs mutations in capreomycin resistance and examined the molecular basis of cross-resistance to the four drugs in M. tuberculosis laboratory-generated mutants and clinical isolates. Spontaneous mutants were generated to the drugs singularly and in combination by plating on medium containing one or two drugs. The frequencies of recovery of the mutants on single- and dual-drug plates were consistent with single-step mutations. The rrs genes of all mutants were sequenced, and the tlyA genes were sequenced for mutants selected on capreomycin, viomycin, or both; MICs of all four drugs were determined. Three rrs mutations (A1401G, C1402T, and G1484T) were found, and each was associated with a particular cross-resistance pattern. Similar mutations and cross-resistance patterns were found in drug-resistant clinical isolates. Overall, the data implicate rrs mutations as a molecular basis for resistance to each of the four drugs. Furthermore, the genotypic and phenotypic differences seen in the development of cross-resistance when M. tuberculosis bacteria were exposed to one or two drugs have implications for selection of treatment regimens.
Topics: Amikacin; Antibiotics, Antitubercular; Capreomycin; Drug Resistance, Bacterial; Genes, rRNA; Humans; Kanamycin; Microbial Sensitivity Tests; Mutation; Mycobacterium tuberculosis; Protein Biosynthesis; RNA, Ribosomal, 16S; Viomycin
PubMed: 16048924
DOI: 10.1128/AAC.49.8.3192-3197.2005 -
Antimicrobial Agents and Chemotherapy Feb 2005Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies...
Capreomycin, an important drug for the treatment of multidrug-resistant tuberculosis, is a macrocyclic peptide antibiotic produced by Saccharothrix mutabolis subspecies capreolus. The basis of resistance to this drug was investigated by isolating and characterizing capreomycin-resistant strains of Mycobacterium smegmatis and Mycobacterium tuberculosis. Colonies resistant to capreomycin were recovered from a library of transposon-mutagenized M. smegmatis. The transposon insertion site of one mutant was mapped to an open reading frame in the unfinished M. smegmatis genome corresponding to the tlyA gene (Rv1694) in the M. tuberculosis H37Rv genome. In M. smegmatis spontaneous capreomycin-resistant mutants, the tlyA gene was disrupted by one of three different naturally occurring insertion elements. Genomic DNAs from pools of transposon mutants of M. tuberculosis H37Rv were screened by PCR by using primers to the tlyA gene and the transposon to detect mutants with an insertion in the tlyA gene. One capreomycin-resistant mutant was recovered that contained the transposon inserted at base 644 of the tlyA gene. Complementation with the wild-type tlyA gene restored susceptibility to capreomycin in the M. smegmatis and M. tuberculosis tlyA transposon mutants. Mutations were found in the tlyA genes of 28 spontaneous capreomycin-resistant mutants generated from three different M. tuberculosis strains and in the tlyA genes of capreomycin-resistant clinical isolates. In in vitro transcription-translation assays, ribosomes from tlyA mutant but not tlyA(+) strains resist capreomycin inhibition of transcription-translation. Therefore, TlyA appears to affect the ribosome, and mutation of tlyA confers capreomycin resistance.
Topics: Antibiotics, Antitubercular; Bacterial Proteins; Blotting, Southern; Capreomycin; Cloning, Molecular; DNA Transposable Elements; DNA, Bacterial; Drug Resistance, Bacterial; Gene Library; Hemolysin Proteins; Humans; Microbial Sensitivity Tests; Mutagenesis; Mutation; Mycobacterium smegmatis; Mycobacterium tuberculosis; Plasmids; Protein Biosynthesis; RNA, Bacterial; RNA, Ribosomal, 16S; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Tuberculosis; Viomycin
PubMed: 15673735
DOI: 10.1128/AAC.49.2.571-577.2005 -
The FEBS Journal Feb 2005Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in...
Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.
Topics: Base Sequence; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli; GTP Pyrophosphokinase; Guanosine Pentaphosphate; RNA, Transfer, Met; Recombinant Proteins; Ribosomes
PubMed: 15670150
DOI: 10.1111/j.1742-4658.2004.04502.x