Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity.
Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics. The modifications involve dehydration of Ser and Thr residues to generate dehydroalanines and dehydrobutyrines, followed by intramolecular attack of cysteines onto the newly formed dehydro amino acids to produce cyclic thioethers. LctM performs both processes during the biosynthesis of lacticin 481. Mutation of the zinc ligands Cys781 and Cys836 to alanine did not affect the dehydration activity of LctM. However, these mutations compromised cyclization activity when investigated with full length or truncated peptide substrates. Mutation of His725, another residue that is fully conserved in lantibiotic cyclases, to Asn resulted in a protein that still catalyzed dehydration of the substrate peptide and also retained cyclization activity, but at a decreased level compared to that of the wild type enzyme. Collectively, these results show that the C-terminal domain of LctM is responsible for cyclization, that the zinc ligands are critical for cyclization, and that dehydration takes place independently from the cyclization activity. Furthermore, these mutant proteins are excellent dehydratases and provide useful tools to investigate the dehydration activity as well as generate dehydrated peptides for study of the cyclization reaction by wild type LctM.
Paul
Moushumi
M
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
Patton
Gregory C
GC
van der Donk
Wilfred A
WA
D001430:Bacteriocins / Q000096:biosynthesis*; D001665:Binding Sites; D003500:Cyclization; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D006836:Hydro-Lyases; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D015032:Zinc / Q000737:chemistry*
D001430:Bacteriocins; D004798:Enzymes; C481644:LctM protein, Lactococcus lactis; D008024:Ligands; C082508:lacticin 481; D006836:Hydro-Lyases; D015032:Zinc