RESUMEN
Acyltransferases (ATs) play an essential role in the polyketide biosynthesis through transferring acyl units into acyl carrier proteins (ACPs) via a self-acylation reaction and a transacylation reaction. Here we used AT10FkbA of FK506 biosynthetic polyketide synthase (PKS) from Streptomyces tsukubaensis YN06 as a model to study the specificity of ATs for acyl units. Our results show that AT10FkbA can form both malonyl-O-AT10FkbA and methylmalonyl-O-AT10FkbA in the self-acylation reaction, however, only malonyl-O-AT10FkbA but not methylmalonyl-O-AT10FkbA can transfer the acyl unit into ACPs in the transacylation reaction. Unlike some ATs that are known to control the acyl specificity in self-acylation reactions, AT10FkbA controls the acyl specificity in transacylation reactions.
Asunto(s)
Proteína Transportadora de Acilo/biosíntesis , Aciltransferasas/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , Proteína Transportadora de Acilo/química , Acilación , Aciltransferasas/química , Secuencia de Aminoácidos , Malonil Coenzima A , Complejos Multienzimáticos , Sintasas Poliquetidas/química , Policétidos/química , Estructura Terciaria de Proteína , Streptomyces/enzimología , Especificidad por Sustrato , Tacrolimus/químicaRESUMEN
The known functions of type II thioesterases (TEIIs) in type I polyketide synthases (PKSs) include selecting of starter acyl units, removal of aberrant extender acyl units, releasing of final products, and dehydration of polyketide intermediates. In this study, we characterized two TEIIs (ScnI and PKSIaTEII) from Streptomyces chattanoogensis L10. Deletion of scnI in S. chattanoogensis L10 decreased the natamycin production by about 43%. Both ScnI and PKSIaTEII could remove acyl units from the acyl carrier proteins (ACPs) involved in the natamycin biosynthesis. Our results show that the TEII could play important roles in both the initiation step and the elongation steps of a polyketide biosynthesis; the intracellular TEIIs involved in different biosynthetic pathways could complement each other.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Natamicina/biosíntesis , Streptomyces/metabolismo , Tioléster Hidrolasas/metabolismo , Secuencia de Aminoácidos , Arginina/metabolismo , Ácido Graso Sintasas/química , Espacio Intracelular/enzimología , Datos de Secuencia Molecular , Streptomyces/citología , Streptomyces/enzimología , Tioléster Hidrolasas/químicaRESUMEN
Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.
Asunto(s)
Proteínas Bacterianas/genética , Dipéptidos/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Evolución Biológica , Magnesio/metabolismo , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismoRESUMEN
Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer, Streptomyces chattanoogensis L10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT in S. chattanoogensis L10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.