Lantibiotic synthetases catalyze the
dehydration of Ser and Thr residues in their
peptide substrates to
dehydroalanine (Dha) and
dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the
thioether cross-links
lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the
dehydratase domain of the best characterized family member,
lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect
dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down
dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net
dehydration: phosphorylation of Ser/Thr residues and the subsequent
phosphate elimination step to form the dehydro
amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased
phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant
enzymes that lacked detectable
dehydration activity. Whereas the Asp242Asn mutant retained
phosphate elimination activity, the Asp259Asn mutant was not able to eliminate
phosphate from a phosphorylated substrate
peptide. A model is presented that accounts for the observed phenotypes of these mutant
enzymes.