Tailoring Enzyme Stringency Masks the Multispecificity of a Lyngbyatoxin (Indolactam Alkaloid) Nonribosomal Peptide Synthetase

Abstract

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. coli, was studied for its amenability towards the biosynthesis of indolactam variants. Modification of culture conditions for our E. 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 in vitro substrate activation by the LtxA NRPS to in vivo product formation. While there was a preference for isoleucine over leucine, the substitution of alternative tailoring domains may unveil the true in vivo effects of the mutations introduced herein.

Gene sdaB Is Involved in the Nematocidal Activity of Enterobacter ludwigii AA4 Against the Pine Wood Nematode Bursaphelenchus xylophilus

“…Until now, little or no data about the contribution of L-serine dehydratase to microbial nematocidal activities has been reported. L-serine is an important biomolecule, and excessive amounts of intracellular serine inhibit the biosynthesis of other amino acids (Zhang et al, 2018(Zhang et al, , 2019Révora et al, 2020;Wang et al, 2020) essential to the production of certain bacterial toxins via the nonribosomal peptide synthetase (NRPS) pathway (Cao et al, 2008;Soeriyadi et al, 2021).…”

Structural advances toward understanding the catalytic activity and conformational dynamics of modular nonribosomal peptide synthetases

“…26,128 Additionally, in the pathway for the indole alkaloid lyngbiatoxin, an MbtH domain is observed fused to a cytochrome P450 in LtxB, an unusual arrangement that may promote protein-protein interactions of the P450 and a partner adenylation domain. 129,130 The MLP family is named aer MbtH, the rst described member that is in the mycobactin pathway of Mycobacterium tuberculosis. 131 Early studies demonstrated that deletion of MLP genes can completely or signicantly compromise the activity of an NRPS pathway.…”

Striving for sustainable biosynthesis: discovery, diversification, and production of antimicrobial drugs in Escherichia coli

“…For example, it was recently demonstrated that fluorinated building blocks can be incorporated into macrolide antibiotics by using engineered polyketide synthases [ 70 , 71 ]. Similarly, the lyngbyatoxin-producing nonribosomal peptide synthetase was engineered towards the production of pharmaceutically relevant indolactam variants [ 64 , 72 , 73 ].…”