RESUMEN
Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.
Asunto(s)
Tereftalatos Polietilenos , Concentración de Iones de Hidrógeno , Tereftalatos Polietilenos/química , Tereftalatos Polietilenos/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Hidrólisis , Temperatura , Modelos MolecularesRESUMEN
BACKGROUND: Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure-function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. RESULTS: We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. CONCLUSIONS: The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.
Asunto(s)
Mycobacterium tuberculosis , Policétidos , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/genética , Ácidos Micólicos/química , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismoRESUMEN
Plastic environmental pollution is a major issue that our generation must face to protect our planet. Plastic recycling has the potential not only to reduce the pollution but also to limit the need for fossil-fuel-based production of new plastics. Enzymes capable of breaking down plastic could thereby support such a circular economy. Polyethylene terephthalate (PET) degrading enzymes have recently attracted considerable interest and have been subjected to intensive enzyme engineering to improve their characteristics. A quadruple mutant of Leaf-branch Compost Cutinase (LCC) was identified as a most efficient and promising enzyme. Here, we use NMR to follow the initial LCC enzyme through its different mutations that lead to its improved performance. We experimentally define the two calcium-binding sites and show their importance on the all-or-nothing thermal unfolding process, which occurs at a temperature of 72°C close to the PET glass transition temperature. Using various NMR probes such as backbone amide, methyl group, and histidine side-chain resonances, we probe the interaction of the enzymes with mono-(2-hydroxyethyl)terephthalic acid. The latter experiments are interpreted in terms of accessibility of the active site to the polymer chain.
Asunto(s)
Plásticos , Tereftalatos Polietilenos , Plásticos/química , TemperaturaRESUMEN
Pks13 is a type I polyketide synthase involved in the final biosynthesis step of mycolic acids, virulence factors, and essential components of the Mycobacterium tuberculosis envelope. Here, we report the biochemical and structural characterization of a 52-kDa fragment containing the acyltransferase domain of Pks13. This fragment retains the ability to load atypical extender units, unusually long chain acyl-CoA with a predilection for carboxylated substrates. High resolution crystal structures were determined for the apo, palmitoylated, and carboxypalmitoylated forms. Structural conservation with type I polyketide synthases and related fatty-acid synthases also extends to the interdomain connections. Subtle changes could be identified both in the active site and in the upstream and downstream linkers in line with the organization displayed by this singular polyketide synthase. More importantly, the crystallographic analysis illustrated for the first time how a long saturated chain can fit in the core structure of an acyltransferase domain through a dedicated channel. The structures also revealed the unexpected binding of a 12-mer peptide that might provide insight into domain-domain interaction.
Asunto(s)
Proteínas Bacterianas/química , Sintasas Poliquetidas/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Proteínas Bacterianas/metabolismo , Unión Competitiva , Dominio Catalítico , Química Farmacéutica/métodos , Clonación Molecular , Cristalografía por Rayos X/métodos , Diseño de Fármacos , Ligandos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
In recent years, enzymatic recycling of the widely used polyester polyethylene terephthalate (PET) has become a complementary solution to current thermomechanical recycling for colored, opaque, and mixed PET. A large set of promising hydrolases that depolymerize PET have been found and enhanced by worldwide initiatives using various methods of protein engineering. Despite the achievements made in these works, it remains difficult to compare enzymes' performance and their applicability to large-scale reactions due to a lack of homogeneity between the experimental protocols used. Here, we pave the way for a standardized enzymatic PET hydrolysis protocol using reaction conditions relevant for larger scale hydrolysis and apply these parameters to four recently reported PET hydrolases (LCCICCG, FAST-PETase, HotPETase, and PES-H1L92F/Q94Y). We show that FAST-PETase and HotPETase have intrinsic limitations that may not permit their application on larger reaction scales, mainly due to their relatively low depolymerization rates. With 80% PET depolymerization, PES-H1L92F/Q94Y may be a suitable candidate for industrial reaction scales upon further rounds of enzyme evolution. LCCICCG outperforms the other enzymes, converting 98% of PET into the monomeric products terephthalic acid (TPA) and ethylene glycol (EG) in 24 h. In addition, we optimized the reaction conditions of LCCICCG toward economic viability, reducing the required amount of enzyme by a factor of 3 and the temperature of the reaction from 72 to 68 °C. We anticipate our findings to advance enzymatic PET hydrolysis toward a coherent assessment of the enzymes and materialize feasibility at larger reaction scales.
RESUMEN
One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4'-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacterium abscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4'-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Coenzima A/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Proteína Transportadora de Acilo/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Coenzima A/química , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Cinética , Mycobacterium abscessus/enzimología , Mycobacterium abscessus/genética , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.
Asunto(s)
Mycobacterium/enzimología , Sintasas Poliquetidas/metabolismo , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Sintasas Poliquetidas/química , Conformación Proteica , Especificidad por SustratoRESUMEN
Mycolic acids (MAs) have a strategic location within the mycobacterial envelope, deeply influencing its architecture and permeability, and play a determinant role in the pathogenicity of mycobacteria. The fatty acid synthase type II (FAS-II) multienzyme system is involved in their biosynthesis. A combination of pull-downs and proteomics analyses led to the discovery of a mycobacterial protein, HadD, displaying highly specific interactions with the dehydratase HadAB of FAS-II. In vitro activity assays and homology modeling showed that HadD is, like HadAB, a hot dog folded (R)-specific hydratase/dehydratase. A hadD knockout mutant of Mycobacterium smegmatis produced only the medium-size alpha'-MAs. Data strongly suggest that HadD is involved in building the third meromycolic segment during the late FAS-II elongation cycles, leading to the synthesis of the full-size alpha- and epoxy-MAs. The change in the envelope composition induced by hadD inactivation strongly altered the bacterial fitness and capacities to aggregate, assemble into colonies or biofilms and spread by sliding motility, and conferred a hypersensitivity to the firstline antimycobacterial drug rifampicin. This showed that the cell surface properties and the envelope integrity were greatly affected. With the alarmingly increasing case number of nontuberculous mycobacterial diseases, HadD appears as an attractive target for drug development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Acido Graso Sintasa Tipo II/metabolismo , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium smegmatis/fisiología , Ácidos Micólicos/metabolismo , Proteínas Bacterianas/genética , Biopelículas , Vías Biosintéticas , Acido Graso Sintasa Tipo II/genética , Eliminación de Gen , Genes Esenciales , Humanos , Mycobacterium smegmatis/genéticaRESUMEN
Enolase is a validated drug target in Trypanosoma brucei. To better characterize its properties and guide drug design efforts, we have determined six new crystal structures of the enzyme, in various ligation states and conformations, and have carried out complementary molecular dynamics simulations. The results show a striking structural diversity of loops near the catalytic site, for which variation can be interpreted as distinct modes of conformational variability that are explored during the molecular dynamics simulations. Our results show that sulfate may, unexpectedly, induce full closure of catalytic site loops whereas, conversely, binding of inhibitor phosphonoacetohydroxamate may leave open a tunnel from the catalytic site to protein surface offering possibilities for drug development. We also present the first complex of enolase with a novel inhibitor 2-fluoro-2-phosphonoacetohydroxamate. The molecular dynamics results further encourage efforts to design irreversible species-specific inhibitors: they reveal that a parasite enzyme-specific lysine may approach the catalytic site more closely than crystal structures suggest and also cast light on the issue of accessibility of parasite enzyme-specific cysteines to chemically modifying reagents. One of the new sulfate structures contains a novel metal-binding site IV within the catalytic site cleft.
Asunto(s)
Fosfopiruvato Hidratasa/química , Trypanosoma brucei brucei/enzimología , Animales , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/químicaRESUMEN
Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28-C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase-ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme.
Asunto(s)
Acilcoenzima A/química , Acilcoenzima A/metabolismo , Hidroliasas/química , Hidroliasas/metabolismo , Mycobacterium tuberculosis/enzimología , Sintasas Poliquetidas/química , Sintasas Poliquetidas/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Mycolate-containing compounds constitute major strategic elements of the protective coat surrounding the tubercle bacillus. We have previously shown that FAAL32-Pks13 polyketide synthase catalyzes the condensation reaction, which produces α-alkyl ß-ketoacids, direct precursors of mycolic acids. In contrast to the current biosynthesis model, we show here that Pks13 catalyzes itself the release of the neosynthesized products and demonstrate that this function is carried by its thioesterase-like domain. Most importantly, in agreement with the prediction of a trehalose-binding pocket in its catalytic site, this domain exhibits an acyltransferase activity and transfers Pks13's products onto an acceptor molecule, mainly trehalose, leading to the formation of the trehalose monomycolate precursor. Thus, this work allows elucidation of the hinge step of the mycolate-containing compound biosynthesis pathway. Above all, it highlights a unique mechanism of transfer of polyketide synthase products in mycobacteria, which is distinct from the conventional intervention of the discrete polyketide-associated protein (Pap)-type acyltransferases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biocatálisis , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/metabolismo , Aciltransferasas/metabolismo , Proteínas Bacterianas/química , Modelos Moleculares , Sintasas Poliquetidas/química , Polímeros/metabolismo , Estructura Terciaria de Proteína , Especificidad por Sustrato , Trehalosa/metabolismoRESUMEN
A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.
Asunto(s)
Venenos de Hormiga/farmacología , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Hormigas/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Ponzoñas/química , Secuencia de Aminoácidos , Animales , Venenos de Hormiga/química , Venenos de Hormiga/aislamiento & purificación , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Espectrometría de Masas en TándemRESUMEN
The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP(Pks13)). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP(Pks13)) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce alpha-alkyl beta-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coenzima A Ligasas/metabolismo , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Sintasas Poliquetidas/metabolismo , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Vías Biosintéticas , Cromatografía Liquida , Coenzima A Ligasas/genética , Electroforesis en Gel de Poliacrilamida , Estructura Molecular , Mycobacterium tuberculosis/genética , Ácidos Micólicos/química , Fragmentos de Péptidos/química , Sintasas Poliquetidas/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en TándemRESUMEN
Mycolic acids are major and specific lipids of Mycobacterium tuberculosis cell envelope. Their synthesis requires the condensation by Pks13 of a C(22)-C(26) fatty acid with the C(50)-C(60) meromycolic acid activated by FadD32, a fatty acyl-AMP ligase essential for mycobacterial growth. A combination of biochemical and enzymatic approaches demonstrated that FadD32 exhibits substrate specificity for relatively long-chain fatty acids. More importantly, FadD32 catalyzes the transfer of the synthesized acyl-adenylate onto specific thioester acceptors, thus revealing the protein acyl-ACP ligase function. Therefore, FadD32 might be the prototype of a group of M. tuberculosis polyketide-synthase-associated adenylation enzymes possessing such activity. A substrate analog of FadD32 inhibited not only the enzyme activity but also mycolic acid synthesis and mycobacterial growth, opening an avenue for the development of novel antimycobacterial agents.
Asunto(s)
Coenzima A Ligasas/metabolismo , Mycobacterium tuberculosis/enzimología , Ácidos Micólicos/metabolismo , Secuencia de Aminoácidos , Coenzima A Ligasas/aislamiento & purificación , Datos de Secuencia Molecular , Ácidos Micólicos/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Dihydroxyacetone-phosphate and phosphonate derivatives were synthesized bearing a N-sulfonyl hydroxamate moiety. The phosphate derivatives represent competitive inhibitors for the class II-FBP aldolase catalyzed reaction, while the phosphonate isosteres are comparatively weaker inhibitors.