RESUMO
Chemical probes capable of reacting with KS (ketosynthase)-bound biosynthetic intermediates were utilized for the investigation of the model typeâ I iterative polyketide synthase 6-methylsalicylic acid synthase (6-MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6-MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6-MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.
Assuntos
Sondas Moleculares , Salicilatos/metabolismo , Cromatografia Líquida de Alta PressãoRESUMO
Correction for 'Novel chemical probes for the investigation of nonribosomal peptide assembly' by Y. T. Candace Ho et al., Chem. Commun., 2017, 53, 7088-7091.
RESUMO
Chemical probes were devised and evaluated for the capture of biosynthetic intermediates involved in the bio-assembly of the nonribosomal peptide echinomycin. Putative intermediate peptide species were isolated and characterised, providing fresh insights into pathway substrate flexibility and paving the way for novel chemoenzymatic approaches towards unnatural peptides.
Assuntos
Equinomicina/biossíntese , Sondas Moleculares/análise , Equinomicina/química , Sondas Moleculares/química , Estrutura MolecularRESUMO
Chemical 'chain termination' probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insights into substrate specificity and in vivo intermediate processing by unusual iterative synthases.
Assuntos
Antibacterianos/biossíntese , Sondas Moleculares/análise , Sondas Moleculares/química , Compostos de Sulfidrila/química , Actinobacteria/metabolismo , Antibacterianos/química , Conformação Molecular , Policetídeo Sintases/metabolismo , Streptomyces/metabolismo , Especificidade por Substrato , Compostos de Sulfidrila/metabolismoRESUMO
Chemical probes capable of reacting with KS (ketosynthase)-bound biosynthetic intermediates were utilized for the investigation of the model typeâ I iterative polyketide synthase 6-methylsalicylic acid synthase (6-MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6-MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6-MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities.
RESUMO
The tripeptide chains of the ergopeptines, a class of pharmacologically important D-lysergic acid alkaloid peptides, are arranged in a unique bicyclic cyclol based on an amino-terminal α-hydroxyamino acid and a terminal orthostructure. D-lysergyl-tripeptides are assembled by the nonribosomal peptide synthetases LPS1 and LPS2 of the ergot fungus Claviceps purpurea and released as N-(D-lysergyl-aminoacyl)-lactams. We show total enzymatic synthesis of ergopeptines catalyzed by a Fe²âº/2-ketoglutarate-dependent dioxygenase (EasH) in conjunction with LPS1/LPS2. Analysis of the reaction indicated that EasH introduces a hydroxyl group into N-(D-lysergyl-aminoacyl)-lactam at α-C of the aminoacyl residue followed by spontaneous condensation with the terminal lactam carbonyl group. Sequence analysis revealed that EasH belongs to the wide and diverse family of the phytanoyl coenzyme A hydroxylases. We provide a high-resolution crystal structure of EasH that is most similar to that of phytanoyl coenzyme A hydroxylase, PhyH, from human.
Assuntos
Dioxigenases/metabolismo , Ergotamina/biossíntese , Ergotamina/química , Ácido Lisérgico/química , Ácido Lisérgico/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Biocatálise , Claviceps/enzimologia , Ciclização , Di-Hidroergotamina/química , Di-Hidroergotamina/metabolismo , Dioxigenases/química , Ergolinas/química , Ergolinas/metabolismo , Humanos , Hidroxilação , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Peptídeo Sintases/metabolismo , Conformação ProteicaRESUMO
In Gram-negative bacteria, the Cpx signal transduction pathway (CpxAR) has an overall regulatory function, but the mechanism of its activation is still poorly understood. Here we investigated the impact of the periplasmic sensor domain of CpxA on signalling by mutational analysis in vitro. Substitutions (R33C and L38P) in the N-terminal region significantly increased CpxA autophosphorylation, whereas a substitution in a predicted beta sheet (E92K), which impaired the inhibitory effect of the auxiliary CpxP protein, had no significant effect on catalytic activity. Thus, our data suggest different regions on the periplasmic domain of CpxA, which impact to modulate signal transduction.