Identification of essential catalytic residues of the cyclase NisC involved in the biosynthesis of nisin.
Nisin is a post-translationally modified antimicrobial peptide that has been widely used in the food industry for several decades. It contains five cyclic thioether cross-links of varying sizes that are installed by a single enzyme, NisC, that catalyzes the addition of cysteines to dehydroamino acids. The recent x-ray crystal structure of NisC has provided the first insights into the catalytic residues responsible for the cyclization step during nisin biosynthesis. In this study, the conserved residues His(212), Arg(280), Asp(141), and Tyr(285) as well as the ligands to the zinc in the active site (Cys(284), Cys(330), and His(331)) were substituted by site-directed mutagenesis. Binding studies showed that all mutants had similar affinities for NisA. Activity assays showed that whereas His(212) and Asp(141) were essential for correct cyclization as judged by the antimicrobial activity of the final product, Arg(280) and Tyr(285) were not. Mutation of zinc ligands to alanine also abolished the enzymatic activity, and these mutant proteins were shown to contain decreased levels of zinc. These results show that the zinc is essential for activity and support a model in which the zinc is used to activate the cysteines in the substrate for nucleophilic attack. These findings also argue against an essential role of Arg(280) and Tyr(285) as an active site general acid/base in the mechanism of cyclization.
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Bo
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Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
van der Donk
Wilfred A
WA
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D019753:Intramolecular Lyases / Q000737:chemistry* / Q000235:genetics*; D008024:Ligands; D008956:Models, Chemical; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009561:Nisin / Q000096:biosynthesis* / Q000737:chemistry; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
D029968:Escherichia coli Proteins; D008024:Ligands; D009561:Nisin; D019753:Intramolecular Lyases; C522741:NisC protein, E coli; D015032:Zinc