RESUMEN
Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Carboxipeptidasas , Humanos , Carboxipeptidasas/química , Peptidil-Dipeptidasa A , Ciclismo , Péptidos/farmacología , Angiotensina II , Fragmentos de PéptidosRESUMEN
Early-stage intermediates in the biosynthesis of erythromycin A by Saccharopolyspora erythraea were intercepted by malonyl carba(dethia)-N-acetyl cysteamines, generated in vivo from the hydrolysis of the corresponding methyl esters.
Asunto(s)
Cisteamina/química , Cisteamina/metabolismo , Eritromicina/biosíntesis , Sintasas Poliquetidas/metabolismo , Biocatálisis , Saccharopolyspora/enzimologíaRESUMEN
The protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and we show that RkD is the enzyme required for RK-682 formation from acyl carrier protein-bound substrates.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Saccharopolyspora/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/química , Estructura Molecular , Familia de Multigenes , Fosfoproteínas Fosfatasas/biosíntesis , Fosfoproteínas Fosfatasas/química , Saccharopolyspora/química , Saccharopolyspora/genética , Especificidad por SustratoRESUMEN
The biosynthetic gene cluster for tetronomycin (TMN), a polyether ionophoric antibiotic that contains four different types of ring, including the distinctive tetronic acid moiety, has been cloned from Streptomyces sp. NRRL11266. The sequenced tmn locus (113 234 bp) contains six modular polyketide synthase (PKS) genes and a further 27 open-reading frames. Based on sequence comparison to related biosynthetic gene clusters, the majority of these can be assigned a plausible role in TMN biosynthesis. The identity of the cluster, and the requirement for a number of individual genes, especially those hypothesised to contribute a glycerate unit to the formation of the tetronate ring, were confirmed by specific gene disruption. However, two large genes that are predicted to encode together a multifunctional PKS of a highly unusual type seem not to be involved in this pathway since deletion of one of them did not alter tetronomycin production. Unlike previously characterised polyether PKS systems, oxidative cyclisation appears to take place on the modular PKS rather than after transfer to a separate carrier protein, while tetronate ring formation and concomitant chain release share common mechanistic features with spirotetronate biosynthesis.
Asunto(s)
Antibacterianos/biosíntesis , Furanos/metabolismo , Familia de Multigenes/genética , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Aminoácidos , Antibacterianos/química , Carbono/metabolismo , Proteínas Portadoras/metabolismo , Clonación Molecular , Éteres/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Estructura Terciaria de Proteína/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Streptomyces/enzimologíaRESUMEN
Ionophoric polyethers are produced by the exquisitely stereoselective oxidative cyclization of a linear polyketide, probably via a triepoxide intermediate. We report here that deletion of either or both of the monBI and monBII genes from the monensin biosynthetic gene cluster gave strains that produced, in place of monensins A and B, a mixture of C-3-demethylmonensins and a number of minor components, including C-9-epi-monensin A. All the minor components were efficiently converted into monensins by subsequent acid treatment. These data strongly suggest that epoxide ring opening and concomitant polyether ring formation are catalyzed by the MonB enzymes, rather than by the enzyme MonCII as previously thought. Consistent with this, homology modeling shows that the structure of MonB-type enzymes closely resembles the recently determined structure of limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis.
Asunto(s)
Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Genes Bacterianos , Monensina/biosíntesis , Monensina/química , Streptomyces/genética , Streptomyces/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , ADN Bacteriano/genética , Epóxido Hidrolasas/química , Eliminación de Gen , Modelos Moleculares , Datos de Secuencia Molecular , Rhodococcus/enzimología , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Streptomyces/enzimologíaRESUMEN
The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.