RESUMO
Indolactam alkaloids are activators of protein kinase C (PKC) and are of pharmacological interest for the treatment of pathologies involving PKC dysregulation. The marine cyanobacterial nonribosomal peptide synthetase (NRPS) pathway for lyngbyatoxin biosynthesis, which we previously expressed in E.â coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E.â coli heterologous expression host and analysis of pathway products suggested the native lyngbyatoxin pathway NRPS does possess a degree of relaxed specificity. Site-directed mutagenesis of two positions within the adenylation domain (A-domain) substrate-binding pocket was performed, resulting in an alteration of substrate preference between valine, isoleucine, and leucine. We observed relative congruence of inâ vitro substrate activation by the LtxA NRPS to inâ vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true inâ vivo effects of the mutations introduced herein.
Assuntos
Toxinas de Lyngbya/biossíntese , Peptídeo Sintases/metabolismo , Toxinas de Lyngbya/química , Estrutura Molecular , Mutagênese Sítio-Dirigida , Peptídeo Sintases/genéticaRESUMO
Paralytic shellfish toxins (PSTs) are neurotoxic alkaloids produced by freshwater cyanobacteria and marine dinoflagellates. Due to their antagonism of voltage-gated sodium channels in excitable cells, certain analogues are of significant pharmacological interest. The biosynthesis of the parent compound, saxitoxin, is initiated with the formation of 4-amino-3-oxo-guanidinoheptane (ethyl ketone) by an unusual polyketide synthase-like enzyme, SxtA. We have heterologously expressed SxtA from Raphidiopsis raciborskii T3 in Escherichia coli and analysed its activity inâ vivo. Ethyl ketone and a truncated analogue, methyl ketone, were detected by HPLC-ESI-HRMS analysis, thus suggesting that SxtA has relaxed substrate specificity inâ vivo. The chemical structures of these products were further verified by tandem mass spectrometry and labelled-precursor feeding with [guanidino-15 N2 ] arginine and [1,2-13 C2 ] acetate. These results indicate that the reactions catalysed by SxtA could give rise to multiple PST variants, including analogues of ecological and pharmacological significance.
Assuntos
Cylindrospermopsis/metabolismo , Escherichia coli/metabolismo , Venenos/metabolismo , Saxitoxina/metabolismo , Canais de Sódio Disparados por Voltagem/química , Cylindrospermopsis/genética , Escherichia coli/genética , Saxitoxina/genética , Especificidade por SubstratoRESUMO
Covering: up to 2018 Marine and freshwater cyanobacteria produce a variety of toxic compounds that pose a threat to the health of humans, livestock and natural ecosystems world-wide. Significant research efforts have been directed towards understanding the biosynthesis of these cyanotoxins in an attempt to reduce their deleterious effects on water quality and, more recently, to harness their biotechnological potential. While a variety of complementary methods (such as bioinformatic analyses and isotope feeding studies) have been employed over the last three decades to address knowledge gaps in this field, this review focuses on the utility of heterologous expression and biochemical studies, including emerging technologies for engineering and expressing complete cyanotoxin gene clusters.