Mechanistic Investigations of the Dehydration Reaction of Lacticin 481 Synthetase Using Site-Directed Mutagenesis#
| Autor: | Young Ok You, Wilfred A. van der Donk |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2007 |
| Předmět: |
Mutant
Molecular Sequence Data Biology Arginine Biochemistry Article Substrate Specificity chemistry.chemical_compound Bacteriocins Dehydroalanine Histidine Amino Acid Sequence Desiccation Phosphorylation Site-directed mutagenesis Lanthionine chemistry.chemical_classification Aspartic Acid Lysine Mutagenesis Lantibiotics Amino acid Enzymes chemistry Models Chemical Dehydratase Mutagenesis Site-Directed Tyrosine |
| Popis: | Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dha), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether crosslinks lanthionine and methyllanthionine, respectively. In this study ten conserved residues have been 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 a mutant 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 lead 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|