RESUMO
Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.
Assuntos
Carbapenêmicos/biossíntese , Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologia , Tienamicinas/biossíntese , Vitamina B 12/metabolismo , Sítios de Ligação , Biocatálise , Coenzimas/metabolismo , Cristalografia por Raios X , Cinética , Metilação , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Streptomyces/metabolismo , Inibidores de beta-Lactamases/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation, and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual ß-lactam-forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for ß-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the ß-lactone-forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized ß-lactam-forming biosynthetic gene clusters by genome mining.
RESUMO
Complex carbapenems are important clinical antibiotics used to treat recalcitrant infections. Their biosynthetic gene clusters contain three essential B12-dependent radical S-adenosylmethionine (rSAM) enzymes. The majority of characterized enzymes in this subfamily catalyze methyl transfer, but only one is required to sequentially install all methionine-derived carbons in complex carbapenems. Therefore, it is probable that the other two rSAM enzymes have noncanonical functions. Through a series of fermentation and in vitro experiments, we show that ThnL uses radical SAM chemistry to catalyze thioether bond formation between C2 of a carbapenam precursor and pantetheine, uniting initial bicycle assembly common to all carbapenems with later tailoring events unique to complex carbapenems. ThnL also catalyzes reversible thiol/disulfide redox on pantetheine. Neither of these functions has been observed previously in a B12-dependent radical SAM enzyme. ThnL expands the known activity of this subclass of enzymes beyond carbon-carbon bond formation or rearrangement. It is also the only radical SAM enzyme currently known to catalyze carbon-sulfur bond formation with only an rSAM Fe-S cluster and no additional auxiliary clusters.
Assuntos
Carbapenêmicos , Proteínas Ferro-Enxofre , S-Adenosilmetionina , Vitamina B 12 , Carbapenêmicos/biossíntese , Carbapenêmicos/química , Carbono , Proteínas Ferro-Enxofre/química , Panteteína/química , S-Adenosilmetionina/química , Sulfetos , Vitamina B 12/químicaRESUMO
Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line-like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of ß-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded ß-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique ß-lactam-synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the ß-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both ß-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between ß-lactam synthesis and allied C domains associated with the appearance of á´ -amino acid and dehydroamino acid residues in other NRPS-derived natural products.
Assuntos
Antibacterianos/biossíntese , Evolução Molecular , Lactamas/metabolismo , Peptídeo Sintases/genética , Histidina/metabolismo , Peptídeo Sintases/metabolismo , Tioléster Hidrolases/metabolismoRESUMO
The carbapenem family of ß-lactam antibiotics displays a remarkably broad spectrum of bactericidal activity, exemplified by meropenem's phase II clinical trial success in patients with pulmonary tuberculosis, a devastating disease for which ß-lactam drugs historically have been notoriously ineffective. The discovery and validation of l,d-transpeptidases (Ldts) as critical drug targets of bacterial cell-wall biosynthesis, which are only potently inhibited by the carbapenem and penem structural classes, gave an enzymological basis for the effectiveness of the first antitubercular ß-lactams. Decades of study have delineated mechanisms of ß-lactam inhibition of their canonical targets, the penicillin-binding proteins; however, open questions remain regarding the mechanisms of Ldt inhibition that underlie programs in drug design, particularly the optimization of kinetic behavior and potency. We have investigated critical features of mycobacterial Ldt inhibition and demonstrate here that the covalent inhibitor meropenem undergoes both reversible reaction and nonhydrolytic off-loading reactions from the cysteine transpeptidase LdtMt2 through a high-energy thioester adduct. Next-generation carbapenem optimization strategies should minimize adduct loss from unproductive mechanisms of Ldt adducts that reduce effective drug concentration.
Assuntos
Antibacterianos/farmacologia , Meropeném/farmacologia , Peptidil Transferases/metabolismo , Antibacterianos/química , Lactonas/química , Lactonas/farmacologia , Meropeném/química , Testes de Sensibilidade MicrobianaRESUMO
The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.
Assuntos
Antraquinonas , Enedi-Inos , Humanos , Antraquinonas/química , Enedi-Inos/química , DNA , Antibióticos Antineoplásicos/químicaRESUMO
Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several inâ vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurolâ S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630â nm, visualized by a prominent color change from blue to orange.
Assuntos
Colorimetria , Peptídeo Sintases , Colorimetria/métodos , Peptídeo Sintases/metabolismo , Ferro , Especificidade por SubstratoRESUMO
Nearly every animal species on Earth contains a unique polyketide synthase (PKS) encoded in its genome, yet no animal-clade PKS has been biochemically characterized, and even the chemical products of these ubiquitous enzymes are known in only a few cases. The earliest animal genome-encoded PKS gene to be identified was SpPks1 from sea urchins. Previous genetic knockdown experiments implicated SpPks1 in synthesis of the sea urchin pigment echinochrome. Here, we express and purify SpPks1, performing biochemical experiments to demonstrate that the sea urchin protein is responsible for the synthesis of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (ATHN). Since ATHN is a plausible precursor of echinochromes, this result defines a biosynthetic pathway to the ubiquitous echinoderm pigments and rewrites the previous hypothesis for echinochrome biosynthesis. Truncation experiments showed that, unlike other type I iterative PKSs so far characterized, SpPks1 produces the naphthalene core using solely ketoacylsynthase (KS), acyltransferase, and acyl carrier protein domains, delineating a unique class of animal nonreducing aromatic PKSs (aPKSs). A series of amino acids in the KS domain define the family and are likely crucial in cyclization activity. Phylogenetic analyses indicate that SpPks1 and its homologs are widespread in echinoderms and their closest relatives, the acorn worms, reinforcing their fundamental importance to echinoderm biology. While the animal microbiome is known to produce aromatic polyketides, this work provides biochemical evidence that animals themselves also harbor ancient, convergent, dedicated pathways to carbocyclic aromatic polyketides. More fundamentally, biochemical analysis of SpPks1 begins to define the vast and unexplored biosynthetic space of the ubiquitous animal PKS family.
Assuntos
Policetídeo Sintases , Policetídeos , Animais , Naftalenos , Filogenia , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Ouriços-do-Mar/metabolismoRESUMO
Mycobacteroides abscessus (Mab) is an emerging environmental microbe that causes chronic lung disease in patients with compromised lung function such as cystic fibrosis and bronchiectasis. It is intrinsically resistant to most antibiotics, therefore there are only few antibiotics that can be repurposed to treat Mab disease. Although current recommendations require daily intake of multiple antibiotics for more than a year, cure rate is low and often associated with significant adverse events. Here, we describe in vivo efficacy of T405, a recently discovered ß-lactam antibiotic of the penem subclass, in a mouse model of pulmonary Mab infection. Imipenem, one of the standard-of-care drugs to treat Mab disease, and also a ß-lactam antibiotic from a chemical class similar to T405, was included as a comparator. Probenecid was included with both T405 and imipenem to reduce the rate of their renal clearance. T405 exhibited bactericidal activity against Mab from the onset of treatment and reduced Mab lung burden at a rate similar to that exhibited by imipenem. The MIC of T405 against Mab was unaltered after 4 weeks of exposure to T405 in the lungs of mice. Using an in vitro assay, we also demonstrate that T405 in combination with imipenem, cefditoren or avibactam exhibits synergism against Mab. Additionally, we describe a scheme for synthesis and purification of T405 on an industrial scale. These attributes make T405 a promising candidate for further preclinical assessment to treat Mab disease.
Assuntos
Imipenem , Infecções por Mycobacterium não Tuberculosas , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Cefalosporinas , Humanos , Imipenem/farmacologia , Imipenem/uso terapêutico , Meropeném/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , beta-Lactamas/uso terapêuticoRESUMO
Non-ribosomal peptide synthetases are giant enzymes composed of modules that house repeated sets of functional domains, which select, activate and couple amino acids drawn from a pool of nearly 500 potential building blocks. The structurally and stereochemically diverse peptides generated in this manner underlie the biosynthesis of a large sector of natural products. Many of their derived metabolites are bioactive such as the antibiotics vancomycin, bacitracin, daptomycin and the ß-lactam-containing penicillins, cephalosporins and nocardicins. Penicillins and cephalosporins are synthesized from a classically derived non-ribosomal peptide synthetase tripeptide (from δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase). Here we report an unprecedented non-ribosomal peptide synthetase activity that both assembles a serine-containing peptide and mediates its cyclization to the critical ß-lactam ring of the nocardicin family of antibiotics. A histidine-rich condensation domain, which typically performs peptide bond formation during product assembly, also synthesizes the embedded four-membered ring. We propose a mechanism, and describe supporting experiments, that is distinct from the pathways that have evolved to the three other ß-lactam antibiotic families: penicillin/cephalosporins, clavams and carbapenems. These findings raise the possibility that ß-lactam rings can be regio- and stereospecifically integrated into engineered peptides for application as, for example, targeted protease inactivators.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Lactamas/química , Lactamas/metabolismo , Peptídeo Sintases/metabolismo , beta-Lactamas/química , beta-Lactamas/metabolismo , Biocatálise , Vias Biossintéticas , Ciclização , Histidina , Serina/metabolismoRESUMO
Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.
Assuntos
Colletotrichum/genética , Genes Fúngicos/genética , Família Multigênica/genética , Perileno/análogos & derivados , DNA Fúngico/genética , Proteínas Fúngicas/genética , Malus/microbiologia , Perileno/metabolismo , Doenças das Plantas/microbiologiaRESUMO
The biosynthesis of the three structural subclasses of enediyne antitumor antibiotics remains largely unknown beyond a common C16 -hexaene precursor. For the anthraquinone-fused subtype, however, an unexpected iodoanthracene γ-thiolactone was established to be a mid-pathway intermediate to dynemicin A. Having deleted a putative flavin-dependent oxidoreductase from the dynemicin biosynthetic gene cluster, we can now report four metabolites that incorporate the iodoanthracene and reveal the formation of the C-N bond linking the anthraquinone and enediyne halves emblematic of this structural subclass. The coupling of an aryl iodide and an amine is familiar from organometallic chemistry, but has little or no precedent in natural product biosynthesis. These metabolites suggest further that enediyne formation occurs early in the overall biosynthesis, and that even earlier events might convert the C16 -hexaene to a common C15 intermediate that partitions to enediyne and anthraquinone building blocks for the heterodimerization.
Assuntos
Antraquinonas/química , Antraquinonas/metabolismo , Enedi-Inos/química , Enedi-Inos/metabolismo , Micromonospora/metabolismo , Micromonospora/genética , Família Multigênica/genética , MutaçãoRESUMO
The N-sulfonated monocyclic ß-lactam ring characteristic of the monobactams confers resistance to zinc metallo-ß-lactamases and affords the most effective class to combat carbapenem-resistant enterobacteria (CRE). Here we report unprecedented nonribosomal peptide synthetase activities, wherein an assembled tripeptide is N-sulfonated in trans before direct synthesis of the ß-lactam ring in a noncanonical, cysteine-containing thioesterase domain. This means of azetidinone synthesis is distinct from the three others known in nature.
Assuntos
Antibacterianos/biossíntese , Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Monobactamas/biossíntese , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/metabolismo , Pseudomonas/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Domínio Catalítico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Monobactamas/química , Monobactamas/farmacologia , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Peptídeo Sintases/genética , EstereoisomerismoRESUMO
Polyketide synthases (PKSs) are microbial multienzymes for the biosynthesis of biologically potent secondary metabolites. Polyketide production is initiated by the loading of a starter unit onto an integral acyl carrier protein (ACP) and its subsequent transfer to the ketosynthase (KS). Initial substrate loading is achieved either by multidomain loading modules or by the integration of designated loading domains, such as starter unit acyltransferases (SAT), whose structural integration into PKS remains unresolved. A crystal structure of the loading/condensing region of the nonreducing PKS CTB1 demonstrates the ordered insertion of a pseudodimeric SAT into the condensing region, which is aided by the SAT-KS linker. Cryo-electron microscopy of the post-loading state trapped by mechanism-based crosslinking of ACP to KS reveals asymmetry across the CTB1 loading/-condensing region, in accord with preferential 1:2 binding stoichiometry. These results are critical for re-engineering the loading step in polyketide biosynthesis and support functional relevance of asymmetric conformations of PKSs.
Assuntos
Proteína de Transporte de Acila/química , Policetídeo Sintases/química , Ascomicetos/metabolismo , Domínio Catalítico , Reagentes de Ligações Cruzadas/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Escherichia coli/metabolismo , Panteteína/química , Fosforilação , Propionatos/química , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Especificidade por SubstratoRESUMO
Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-ß-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-ß-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-ß-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.
Assuntos
Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Biomimética , Mutagênese Sítio-Dirigida , Panteteína/isolamento & purificação , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeos/química , Conformação ProteicaRESUMO
Bacterial survival requires an intact peptidoglycan layer, a three-dimensional exoskeleton that encapsulates the cytoplasmic membrane. Historically, the final steps of peptidoglycan synthesis are known to be carried out by D,D-transpeptidases, enzymes that are inhibited by the ß-lactams, which constitute >50% of all antibacterials in clinical use. Here, we show that the carbapenem subclass of ß-lactams are distinctly effective not only because they inhibit D,D-transpeptidases and are poor substrates for ß-lactamases, but primarily because they also inhibit non-classical transpeptidases, namely the L,D-transpeptidases, which generate the majority of linkages in the peptidoglycan of mycobacteria. We have characterized the molecular mechanisms responsible for inhibition of L,D-transpeptidases of Mycobacterium tuberculosis and a range of bacteria including ESKAPE pathogens, and used this information to design, synthesize and test simplified carbapenems with potent antibacterial activity.
Assuntos
Antibacterianos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Peptidil Transferases/antagonistas & inibidores , beta-Lactamas/farmacologia , Antibacterianos/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Peptidil Transferases/metabolismo , Relação Estrutura-Atividade , beta-Lactamas/químicaRESUMO
Modular nonribosomal peptide synthetases (NRPSs) are large, multidomain engines of bioactive natural product biosynthesis that function as molecular "assembly lines" in which monomer units are selectively bound, modified, and linked in a specific order and number dictated by their mega-enzyme templates. Recently, a condensation domain in an NRPS was discovered to carry out the synthesis of an integrated ß-lactam ring from a substrate seryl residue during antibiotic biosynthesis. We report here a series of experiments supporting a mechanism that involves C-N bond formation by stepwise elimination/addition reactions followed by canonical NRPS-catalyzed amide bond synthesis to achieve ß-lactam formation. Partitioning of reactive intermediates formed during the multistep catalytic cycle provided insight into the ability of the NRPS to overcome the reversibility of corresponding reactions in solution and enforce directionality during synthesis.
Assuntos
Antibacterianos/biossíntese , Peptídeo Sintases/metabolismo , beta-Lactamas/metabolismo , Antibacterianos/química , Biocatálise , Domínio Catalítico , Conformação Molecular , beta-Lactamas/químicaRESUMO
Covering: up to mid of 2018 Type I fatty acid synthases (FASs) are giant multienzymes catalyzing all steps of the biosynthesis of fatty acids from acetyl- and malonyl-CoA by iterative precursor extension. Two strikingly different architectures of FAS evolved in yeast (as well as in other fungi and some bacteria) and metazoans. Yeast-type FAS (yFAS) assembles into a barrel-shaped structure of more than 2 MDa molecular weight. Catalytic domains of yFAS are embedded in an extensive scaffolding matrix and arranged around two enclosed reaction chambers. Metazoan FAS (mFAS) is a 540 kDa X-shaped dimer, with lateral reaction clefts, minimal scaffolding and pronounced conformational variability. All naturally occurring yFAS are strictly specialized for the production of saturated fatty acids. The yFAS architecture is not used for the biosynthesis of any other secondary metabolite. On the contrary, mFAS is related at the domain organization level to major classes of polyketide synthases (PKSs). PKSs produce a variety of complex and potent secondary metabolites; they either act iteratively (iPKS), or are linked via directed substrate transfer into modular assembly lines (modPKSs). Here, we review the architectures of yFAS, mFAS, and iPKSs. We rationalize the evolution of the yFAS assembly, and provide examples for re-engineering of yFAS. Recent studies have provided novel insights into the organization of iPKS. A hybrid crystallographic model of a mycocerosic acid synthase-like Pks5 yielded a comprehensive visualization of the organization and dynamics of fully-reducing iPKS. Deconstruction experiments, structural and functional studies of specialized enzymatic domains, such as the product template (PT) and the starter-unit acyltransferase (SAT) domain have revealed functional principles of non-reducing iterative PKS (NR-PKSs). Most recently, a six-domain loading region of an NR-PKS has been visualized at high-resolution together with cryo-EM studies of a trapped loading intermediate. Altogether, these data reveal the related, yet divergent architectures of mFAS, iPKS and also modPKSs. The new insights highlight extensive dynamics, and conformational coupling as key features of mFAS and iPKS and are an important step towards collection of a comprehensive series of snapshots of PKS action.
Assuntos
Ácido Graxo Sintase Tipo I/química , Policetídeo Sintases/química , Aciltransferases/química , Aciltransferases/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Evolução Molecular , Ácido Graxo Sintase Tipo I/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Filogenia , Policetídeo Sintases/metabolismo , Conformação Proteica , Metabolismo Secundário , Leveduras/enzimologiaRESUMO
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 ß-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.
Assuntos
Carbapenêmicos/química , Metilação de DNA , Tienamicinas/biossíntese , Antibacterianos/química , Catálise , Cefalosporinas/química , Cromatografia Líquida , Clonagem Molecular , Desenho de Fármacos , Escherichia coli , Fermentação , Metilação , Penicilinas/química , S-Adenosilmetionina/química , Streptomyces , Espectrometria de Massas em Tandem , Tienamicinas/química , Vitamina B 12/química , beta-Lactamas/químicaRESUMO
Despite the identification of a ß-hydroxyhexaene produced by the enediyne polyketide synthases (PKSs), the post-PKS biosynthetic steps to the individual members of this antitumor and antibiotic family remain largely unknown. The massive biosynthetic gene clusters (BGCs) that direct the formation of each product caution that many steps could be required. It was recently demonstrated that the enediyne PKS in the dynemicinâ A BGC from Micromonospora chersina gives rise to both the anthraquinone and enediyne halves of the molecule. We now present the first evidence for a mid-pathway intermediate in dynemicinâ A biosynthesis, an iodoanthracene bearing a fused thiolactone, which was shown to be incorporated selectively into the final product. This unusual precursor reflects just how little is understood about these biosynthetic pathways, yet constrains the mechanisms that can act to achieve the key heterodimerization to the anthraquinone-containing subclass of enediynes.