RESUMEN
There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.
Asunto(s)
Actinomycetales/enzimología , Antibacterianos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Metiltransferasas/química , Metiltransferasas/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Antibacterianos/química , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , Metiltransferasas/genética , Modelos Moleculares , Mutación Puntual , Unión Proteica , Multimerización de Proteína , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Teicoplanina/metabolismoRESUMEN
Mutants capable of utilizing the pyrimidine biosynthetic intermediates carbamoylaspartate and dihydroorotate for growth were derived from pyrimidine auxotrophs of Salmonella enterica serovar Typhimurium LT2. The gain-of-function phenotypes both resulted from mutations in a single gene, yhiT, the third gene of a putative four-gene operon, yhiVUTS, for which there is no homologous region in Escherichia coli. Notably, when a mutant yhiT allele was transferred to a pyrimidine-requiring E. coli strain, the transformant was then capable of using carbamoylaspartate or dihydrorotate as a pyrimidine source. The operon arrangement of the yhiVUTS genes was supported by genetic analyses and studies employing RT-PCR, coupled to the determination of the transcriptional start site using 5'-random amplification of cDNA ends (RACE). Computer-generated predictions indicated that YhiT is an integral membrane protein with 12 putative transmembrane domains typical of bacterial transport proteins. Competition experiments showed that mutant YhiT interacts with the C4-dicarboxylates succinate and malate, as well as the amino acids aspartate and asparagine. The native function of wild-type YhiT remains undetermined, but the collective results are consistent with a role as a general transporter of C4-dicarboxylates and other compounds with a similar basic structure.
Asunto(s)
Ácido Aspártico/análogos & derivados , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Mutación , Ácido Orótico/análogos & derivados , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Malatos/metabolismo , Datos de Secuencia Molecular , Operón , Ácido Orótico/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmonella typhimurium/genética , Ácido Succínico/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vancomycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934, a unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurally characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys(12), His(67), and Ser(98)), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp(34), His(43), Phe(77), Trp(132), and Glu(205).
Asunto(s)
Antibacterianos/química , Proteínas Bacterianas/química , Modelos Moleculares , Ristocetina/análogos & derivados , Streptomyces/enzimología , Sulfotransferasas/química , Secuencias de Aminoácidos , Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Dimerización , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ristocetina/biosíntesis , Ristocetina/química , Sulfotransferasas/metabolismo , Teicoplanina/biosíntesis , Teicoplanina/químicaRESUMEN
The unique glycopeptide antibiotic A47934, produced by Streptomyces toyocaensis, possesses a nonglycosylated heptapeptide core that is sulfated on the phenolic hydroxyl of the N-terminal 4-hydroxy-L-phenylglycine residue. Genetic and biochemical experiments confirmed that StaL is a sulfotransferase capable of sulfating the predicted crosslinked heptapeptide substrate to produce A47934 both in vivo and in vitro. Incubation of purified His(6)-StaL with various substrates in vitro revealed substrate specificity and yielded two sulfo-glycopeptide antibiotics: sulfo-teicoplanin aglycone and sulfo-teicoplanin. Quantification of the antibacterial activity of desulfo-A47934, A47934, teicoplanin, and sulfo-teicoplanin demonstrated that sulfation slightly increased the minimum inhibitory concentration. This unique modification by sulfation expands glycopeptide diversity with potential application for the development of new antibiotics.