Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis.
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.
You
Young Ok
YO
Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
van der Donk
Wilfred A
WA
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D001430:Bacteriocins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D003890:Desiccation; D004798:Enzymes / Q000737:chemistry / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008239:Lysine / Q000235:genetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics
D001430:Bacteriocins; D004798:Enzymes; C481644:LctM protein, Lactococcus lactis; C082508:lacticin 481; D001224:Aspartic Acid; D014443:Tyrosine; D006639:Histidine; D001120:Arginine; D008239:Lysine