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J. Biol. Chem., Vol. 282, Issue 29, 21169-21175, July 20, 2007

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Identification of Essential Catalytic Residues of the Cyclase NisC Involved in the Biosynthesis of Nisin^*

Bo Li and Wilfred A. van der Donk¹

From the Departments of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

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 His²¹², Arg²⁸⁰, Asp¹⁴¹, and Tyr²⁸⁵ as well as the ligands to the zinc in the active site (Cys²⁸⁴, Cys³³⁰, and His³³¹) were substituted by site-directed mutagenesis. Binding studies showed that all mutants had similar affinities for NisA. Activity assays showed that whereas His²¹² and Asp¹⁴¹ were essential for correct cyclization as judged by the antimicrobial activity of the final product, Arg²⁸⁰ and Tyr²⁸⁵ 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 Arg²⁸⁰ and Tyr²⁸⁵ as an active site general acid/base in the mechanism of cyclization.

Received for publication, March 1, 2007 , and in revised form, May 16, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

¹ To whom correspondence should be addressed: Depts. of Biochemistry and Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801. Tel.: 217-244-5360; Fax: 217-244-8024; E-mail: vddonk{at}uiuc.edu.

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