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Craig A Townsend - One of the best experts on this subject based on the ideXlab platform.
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Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in Thienamycin biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Daniel R. Marous, Evan P. Lloyd, Andrew R. Buller, Kristos A. Moshos, Tyler L. Grove, Anthony J. Blaszczyk, Squire J Booker, Craig A TownsendAbstract:Despite their broad anti-infective utility, the biosynthesis of the paradigm carbapenem antibiotic, Thienamycin, remains largely unknown. Apart from the first two steps shared with a simple carbapenem, the pathway sharply diverges to the more structurally complex members of this class of β-lactam antibiotics, such as Thienamycin. Existing evidence points to three putative cobalamin-dependent radical S-adenosylmethionine (RS) enzymes, ThnK, ThnL, and ThnP, as potentially being responsible for assembly of the ethyl side chain at C6, bridgehead epimerization at C5, installation of the C2-thioether side chain, and C2/3 desaturation. The C2 substituent has been demonstrated to be derived by stepwise truncation of CoA, but the timing of these events with respect to C2–S bond formation is not known. We show that ThnK of the three apparent cobalamin-dependent RS enzymes performs sequential methylations to build out the C6-ethyl side chain in a stereocontrolled manner. This enzymatic reaction was found to produce expected RS methylase coproducts S-adenosylhomocysteine and 5′-deoxyadenosine, and to require cobalamin. For double methylation to occur, the carbapenam substrate must bear a CoA-derived C2-thioether side chain, implying the activity of a previous sulfur insertion by an as-yet unidentified enzyme. These insights allow refinement of the central steps in complex carbapenem biosynthesis.
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definition of the common and divergent steps in carbapenem β lactam antibiotic biosynthesis
ChemBioChem, 2011Co-Authors: Micah J Bodner, Evan P. Lloyd, Kristos A. Moshos, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of Thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to Thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.
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non heme iron oxygenases generate natural structural diversity in carbapenem antibiotics
Journal of the American Chemical Society, 2010Co-Authors: Micah J Bodner, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the Thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl Thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.
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Four enzymes define the incorporation of coenzyme A in Thienamycin biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Michael F Freeman, Kristos A. Moshos, Micah J Bodner, Rongfeng Li, Craig A TownsendAbstract:The enzymatic activities of three proteins encoded by the Thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the β-lactam antibiotic Thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible Thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylThienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, Thienamycin and N-acetylThienamycin, in a process likely to be general for the broader class of these antibiotics.
Gloria Blanco - One of the best experts on this subject based on the ideXlab platform.
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activation and silencing of secondary metabolites in streptomyces albus and streptomyces lividans after transformation with cosmids containing the Thienamycin gene cluster from streptomyces cattleya
Archives of Microbiology, 2014Co-Authors: Alfredo F Brana, Miriam Rodriguez, Pallab Pahari, Jurgen Rohr, Luis A Garcia, Gloria BlancoAbstract:Activation and silencing of antibiotic production was achieved in Streptomyces albus J1074 and Streptomyces lividans TK21 after introduction of genes within the Thienamycin cluster from S. cattleya. Dramatic phenotypic and metabolic changes, involving activation of multiple silent secondary metabolites and silencing of others normally produced, were found in recombinant strains harbouring the Thienamycin cluster in comparison to the parental strains. In S. albus, ultra-performance liquid chromatography purification and NMR structural elucidation revealed the identity of four structurally related activated compounds: the antibiotics paulomycins A, B and the paulomenols A and B. Four volatile compounds whose biosynthesis was switched off were identified by gas chromatography–mass spectrometry analyses and databases comparison as pyrazines; including tetramethylpyrazine, a compound with important clinical applications to our knowledge never reported to be produced by Streptomyces. In addition, this work revealed the potential of S. albus to produce many others secondary metabolites normally obtained from plants, including compounds of medical relevance as dihydro-β-agarofuran and of interest in perfume industry as β-patchoulene, suggesting that it might be an alternative model for their industrial production. In S. lividans, actinorhodins production was strongly activated in the recombinant strains whereas undecylprodigiosins were significantly reduced. Activation of cryptic metabolites in Streptomyces species might represent an alternative approach for pharmaceutical drug discovery.
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comparative analysis of a cryptic Thienamycin like gene cluster identified in streptomyces flavogriseus by genome mining
Archives of Microbiology, 2012Co-Authors: Gloria BlancoAbstract:In silico database searches allowed the identification in the S. flavogriseus ATCC 33331 genome of a carbapenem gene cluster highly related to the S. cattleya Thienamycin one. This is the second cluster found for a complex highly substituted carbapenem. Comparative analysis revealed that both gene clusters display a high degree of synteny in gene organization and in protein conservation. Although the cluster appears to be silent under our laboratory conditions, the putative metabolic product was predicted from bioinformatics analyses using sequence comparison tools. These data, together with previous reports concerning epiThienamycins production by S. flavogriseus strains, suggest that the cluster metabolic product might be a Thienamycin-like carbapenem, possibly the epimeric epiThienamycin. This finding might help in understanding the biosynthetic pathway to Thienamycin and other highly substituted carbapenems. It also provides another example of genome mining in Streptomyces sequenced genomes as a powerful approach for novel antibiotic discovery.
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mutational analysis of the Thienamycin biosynthetic gene cluster from streptomyces cattleya
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:The generation of non-Thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in Thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in Thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of Thienamycin production, whereas a 2- to 3-fold increase in Thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for Thienamycin biosynthesis, as Thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the Thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of Thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydroThienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-Thienamycin-producing mutants, lead to a proposal for some enzymatic steps during Thienamycin assembly.
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transcriptional organization of thni regulated Thienamycin biosynthetic genes in streptomyces cattleya
The Journal of Antibiotics, 2010Co-Authors: Miriam Rodriguez, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:Transcriptional organization of ThnI-regulated Thienamycin biosynthetic genes in Streptomyces cattleya
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identification of transcriptional activators for Thienamycin and cephamycin c biosynthetic genes within the Thienamycin gene cluster from streptomyces cattleya
Molecular Microbiology, 2008Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:Summary Two regulatory genes, thnI and thnU, were identified in the Thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished Thienamycin production showing its involvement in Thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for Thienamycin biosynthesis that regulates the expression of nine genes involved in Thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in Thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.
Jose A Salas - One of the best experts on this subject based on the ideXlab platform.
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mutational analysis of the Thienamycin biosynthetic gene cluster from streptomyces cattleya
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:The generation of non-Thienamycin-producing mutants with mutations in the thnL, thnN, thnO, and thnI genes within the thn gene cluster from Streptomyces cattleya and their involvement in Thienamycin biosynthesis and regulation were previously reported. Four additional mutations were independently generated in the thnP, thnG, thnR, and thnT genes by insertional inactivation. Only the first two genes were found to play a role in Thienamycin biosynthesis, since these mutations negatively or positively affect antibiotic production. A mutation of thnP results in the absence of Thienamycin production, whereas a 2- to 3-fold increase in Thienamycin production was observed for the thnG mutant. On the other hand, mutations in thnR and thnT showed that although these genes were previously reported to participate in this pathway, they seem to be nonessential for Thienamycin biosynthesis, as Thienamycin production was not affected in these mutants. High-performance liquid chromatography (HPLC)-mass spectrometry (MS) analysis of all available mutants revealed some putative intermediates in the Thienamycin biosynthetic pathway. A compound with a mass corresponding to carbapenam-3-carboxylic acid was detected in some of the mutants, suggesting that the assembly of the bicyclic nucleus of Thienamycin might proceed in a way analogous to that of the simplest natural carbapenem, 1-carbapen-2-em-3-carboxylic acid biosynthesis. The accumulation of a compound with a mass corresponding to 2,3-dihydroThienamycin in the thnG mutant suggests that it might be the last intermediate in the biosynthetic pathway. These data, together with the establishment of cross-feeding relationships by the cosynthesis analysis of the non-Thienamycin-producing mutants, lead to a proposal for some enzymatic steps during Thienamycin assembly.
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transcriptional organization of thni regulated Thienamycin biosynthetic genes in streptomyces cattleya
The Journal of Antibiotics, 2010Co-Authors: Miriam Rodriguez, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:Transcriptional organization of ThnI-regulated Thienamycin biosynthetic genes in Streptomyces cattleya
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identification of transcriptional activators for Thienamycin and cephamycin c biosynthetic genes within the Thienamycin gene cluster from streptomyces cattleya
Molecular Microbiology, 2008Co-Authors: Miriam Rodriguez, Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Jose A Salas, Gloria BlancoAbstract:Summary Two regulatory genes, thnI and thnU, were identified in the Thienamycin (thn) gene cluster from Streptomyces cattleya. ThnI resembles LysR-type transcriptional activators and ThnU belongs to the SARP family of transcriptional activators. Their functional role was established after independent inactivation by gene replacement together with transcriptional analysis involving reverse transcription polymerase chain reaction (RT-PCR). Deletion of thnI abolished Thienamycin production showing its involvement in Thienamycin biosynthesis. Gene expression analysis applied to the thn gene cluster demonstrated that ThnI is a transcriptional activator essential for Thienamycin biosynthesis that regulates the expression of nine genes involved in Thienamycin assembly and export (thnH, thnJ, thnK, thnL, thnM, thnN, thnO, thnP and thnQ). Unexpectedly, the thnU disrupted mutant was not affected in Thienamycin production but turned out to be essential for cephamycin C biosynthesis. Transcript analysis applied to early and late structural genes for cephamycin C biosynthesis (pcbAB and cmcI), revealed that ThnU is the transcriptional activator of these cephamycin C genes although they are not physically linked to the thn cluster. In addition, it was shown that deletion of thnI has an upregulatory effect on pcbAB and cmcI transcription consistent with a significant increase in cephamycin C biosynthesis in this mutant.
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the biosynthetic gene cluster for the β lactam carbapenem Thienamycin in streptomyces cattleya
Chemistry & Biology, 2003Co-Authors: Luz Elena Nunez, Alfredo F Brana, Carmen Mendez, Gloria Blanco, Jose A SalasAbstract:Abstract β-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic β-lactam antibiotics. This involves the participation of a β-lactam synthetase. Using available information from β-lactam synthetases, we generated a probe for the isolation of the Thienamycin cluster from Streptomyces cattleya . Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of Thienamycin biosynthesis leading to the formation of the β-lactam ring. Other genes necessary for the biosynthesis of Thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in Thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating Thienamycin nonproducing mutants.
Michael F Freeman - One of the best experts on this subject based on the ideXlab platform.
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definition of the common and divergent steps in carbapenem β lactam antibiotic biosynthesis
ChemBioChem, 2011Co-Authors: Micah J Bodner, Evan P. Lloyd, Kristos A. Moshos, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of Thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to Thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.
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non heme iron oxygenases generate natural structural diversity in carbapenem antibiotics
Journal of the American Chemical Society, 2010Co-Authors: Micah J Bodner, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the Thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl Thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.
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Non-Heme Iron Oxygenases Generate Natural Structural Diversity in Carbapenem Antibiotics
2009Co-Authors: Impact Factor, Micah J Bodner, Ryan M Phelan, Doi Source Pubmed, Micah Bodner, Ryan Phelan, Michael Freeman, See Profile, Michael F FreemanAbstract:Carbapenem antibiotics are of clinical importance because of their high potency, broad spectrum of antimicrobial activity, and resistance to most -lactamases.1 Thienamycin (1) (Figure 1), the most potent natural member of this family, co-occurs in Strepto-myces cattleya with four carbapenems that are distinguished by their C-2/C-6 substituents.2 There are more than 50 known carbapenam/ em metabolites, many of which are differentiated only by the oxidation state of their C-2/C-6 substituents. The C-6 ethyl side chain of 1 is derived by C1-donations from methionine3,4 and can be methyl, ethyl, or isopropyl, which can be saturated, unsaturated, hydroxylated, or sulfated. Recent work has established that coenzyme A is successively truncated by three enzymes encoded by the Thienamycin gene cluster to give the C-2 cysteamine moiety.5 This side chain can be pantetheine, but is generally cysteamine, which can be N-acetylated or N-propionylated, desaturated an
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Four enzymes define the incorporation of coenzyme A in Thienamycin biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Michael F Freeman, Kristos A. Moshos, Micah J Bodner, Rongfeng Li, Craig A TownsendAbstract:The enzymatic activities of three proteins encoded by the Thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the β-lactam antibiotic Thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible Thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylThienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, Thienamycin and N-acetylThienamycin, in a process likely to be general for the broader class of these antibiotics.
Micah J Bodner - One of the best experts on this subject based on the ideXlab platform.
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definition of the common and divergent steps in carbapenem β lactam antibiotic biosynthesis
ChemBioChem, 2011Co-Authors: Micah J Bodner, Evan P. Lloyd, Kristos A. Moshos, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Approximately 50 naturally occurring carbapenem β-lactam antibiotics are known. All but one of these have been isolated from Streptomyces species and are disubstituted structural variants of a simple core that is synthesized by Pectobacterium carotovorum (Erwinia carotovora), a phylogenetically distant plant pathogen. While the biosynthesis of the simple carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, is impressively efficient requiring only three enzymes, CarA, CarB and CarC, the formation of Thienamycin, one of the former group of metabolites from Streptomyces, is markedly more complex. Despite their phylogenetic separation, bioinformatic analysis of the encoding gene clusters suggests that the two pathways could be related. Here we demonstrate with gene swapping, stereochemical and kinetics experiments that CarB and CarA and their S. cattleya orthologues, ThnE and ThnM, respectively, are functionally and stereochemically equivalent, although their catalytic efficiencies differ. The biosynthetic pathways, therefore, to Thienamycin, and likely to the other disubstituted carbapenems, and to the simplest carbapenem, (5R)-carbapen-2-em-3-carboxylic acid, are initiated in the same manner, but share only two common steps before diverging.
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non heme iron oxygenases generate natural structural diversity in carbapenem antibiotics
Journal of the American Chemical Society, 2010Co-Authors: Micah J Bodner, Ryan M Phelan, Michael F Freeman, Craig A TownsendAbstract:Carbapenems are a clinically important antibiotic family. More than 50 naturally occurring carbapenam/ems are known and are distinguished primarily by their C-2/C-6 side chains where many are only differentiated by the oxidation states of these substituents. With a limited palette of variations the carbapenem family comprises a natural combinatorial library, and C-2/C-6 oxidation is associated with increased efficacy. We demonstrate that ThnG and ThnQ encoded by the Thienamycin gene cluster in Streptomyces cattleya oxidize the C-2 and C-6 moieties of carbapenems, respectively. ThnQ stereospecifically hydroxylates PS-5 (5) giving N-acetyl Thienamycin (2). ThnG catalyzes sequential desaturation and sulfoxidation of PS-5 (5), giving PS-7 (7) and its sulfoxide (9). The enzymes are relatively substrate selective but are proposed to give rise to the oxidative diversity of carbapenems produced by S. cattleya, and orthologues likely function similarly in allied streptomyces. Elucidating the roles of ThnG and ThnQ will focus further investigations of carbapenem antibiotic biosynthesis.
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Non-Heme Iron Oxygenases Generate Natural Structural Diversity in Carbapenem Antibiotics
2009Co-Authors: Impact Factor, Micah J Bodner, Ryan M Phelan, Doi Source Pubmed, Micah Bodner, Ryan Phelan, Michael Freeman, See Profile, Michael F FreemanAbstract:Carbapenem antibiotics are of clinical importance because of their high potency, broad spectrum of antimicrobial activity, and resistance to most -lactamases.1 Thienamycin (1) (Figure 1), the most potent natural member of this family, co-occurs in Strepto-myces cattleya with four carbapenems that are distinguished by their C-2/C-6 substituents.2 There are more than 50 known carbapenam/ em metabolites, many of which are differentiated only by the oxidation state of their C-2/C-6 substituents. The C-6 ethyl side chain of 1 is derived by C1-donations from methionine3,4 and can be methyl, ethyl, or isopropyl, which can be saturated, unsaturated, hydroxylated, or sulfated. Recent work has established that coenzyme A is successively truncated by three enzymes encoded by the Thienamycin gene cluster to give the C-2 cysteamine moiety.5 This side chain can be pantetheine, but is generally cysteamine, which can be N-acetylated or N-propionylated, desaturated an
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Four enzymes define the incorporation of coenzyme A in Thienamycin biosynthesis
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Michael F Freeman, Kristos A. Moshos, Micah J Bodner, Rongfeng Li, Craig A TownsendAbstract:The enzymatic activities of three proteins encoded by the Thienamycin gene cluster of Streptomyces cattleya (ThnR, ThnH, and ThnT) have been shown to incrementally cleave CoA to afford the active side-chain component of the β-lactam antibiotic Thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible Thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. Finally, a fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya cometabolite, N-acetylThienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, Thienamycin and N-acetylThienamycin, in a process likely to be general for the broader class of these antibiotics.