RESUMO
Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), ß-ureidodehydroalanine, l-capreomycidine, and ß-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.
Assuntos
Aminobutiratos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Capreomicina/biossíntese , Streptomyces/enzimologia , Sítios de Ligação , Catálise , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Especificidade por SubstratoRESUMO
The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.
Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Capreomicina/biossíntese , Família Multigênica/fisiologia , Peptídeo Sintases/metabolismo , Viomicina/biossíntese , Bactérias/genética , Proteínas de Bactérias/genética , Peptídeo Sintases/genéticaRESUMO
Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.
Assuntos
Produtos Biológicos/biossíntese , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeo Sintases/fisiologia , Antibacterianos/biossíntese , Bleomicina/biossíntese , Capreomicina/biossíntese , Domínio Catalítico , Ciclosporina/metabolismo , Glicopeptídeos/biossíntese , Peptídeo Sintases/química , Quinoxalinas/metabolismo , beta-Lactamas/metabolismoRESUMO
Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.
Assuntos
Antibióticos Antituberculose/biossíntese , Antibióticos Antituberculose/farmacologia , Capreomicina/biossíntese , Capreomicina/farmacologia , Genes Bacterianos , Família Multigênica , Actinobacteria/efeitos dos fármacos , Actinobacteria/genética , Actinobacteria/metabolismo , Sequência de Aminoácidos , Antibióticos Antituberculose/química , Sequência de Bases , Capreomicina/química , Conjugação Genética , Primers do DNA/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Resistência a Canamicina/genética , Dados de Sequência Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/genética , Streptomyces lividans/genética , Streptomyces lividans/metabolismoRESUMO
Viomycin capreomycin, antibiotics produced by Streptomyces vinaceus and S. capreolus respectively, are potent inhibitors of bacterial protein synthesis. Although these organisms are highly tolerant of their own products in vivo, their ribosomes are fully sensitive to the action of the drugs in vitro. However, they processes novel, antibiotic-inactivating enzymes (viomycin phosphotransferase, capreomycin phosphotransferase, capreomycin acetyltransferase) which, in addition to possible biosynthetic roles, may contribute to the resistances observed in vivo.