Expanding Substrate Promiscuity by Engineering a Novel Adenylating-Methylating NRPS Bifunctional Enzyme

• Published in: Chembiochem : a European journal of chemical biology Vol. 17; no. 14; pp. 1328 - 1332
• Main Authors: Shrestha, Sanjib K., Garneau-Tsodikova, Sylvie
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 15-07-2016; Wiley Subscription Services, Inc
• Subjects: Adenosine Monophosphate; Amino Acids - metabolism; Biological Products - chemical synthesis; domain swapping; enzyme catalysis; Enzymes; kutzneride; Methylation; natural products; nonribosomal peptides; Peptide Synthases - chemistry; Protein Domains; Protein Engineering - methods; Substrate Specificity; thiocoraline; natural products; enzyme catalysis; thiocoraline; kutzneride; domain swapping; nonribosomal peptides
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201600234

Nonribosomal peptides synthetases (NRPSs), which are multifunctional mega‐enzymes producing many biologically active metabolites, are ideal targets for enzyme engineering. NRPS adenylation domains play a critical role in selecting/activating the amino acids to be transferred to downstream NRPS domains in the biosynthesis of natural products. Both monofunctional and bifunctional A domains interrupted with an auxiliary domain are found in nature. Here, we show that a bifunctional interrupted A domain can be uninterrupted by deleting its methyltransferase auxiliary domain portion to make an active monofunctional enzyme. We also demonstrate that a portion of an auxiliary domain with almost no sequence identity to the original auxiliary domain can be insert into naturally interrupted A domain to develop a new active bifunctional A domain with increased substrate profile. This work shows promise for the creation of new interrupted A domains in engineered NRPS enzymes.
New enzymes developed: By removing the methyltransferase (M) portion of a bifunctional adenylating–methylating enzyme, a novel monofunctional adenylating enzyme was produced. By replacing the M portion of a bifunctional enzyme, a novel bifunctional enzyme with expanded substrate promiscuity was created.