The role of cep15 in the biosynthesis of chloroeremomycin: reactivation of an ancestral catalytic function.

The gene clusters of several glycopeptides contain genes that encode COG2120 domain zinc-dependent N-acetylglucosaminyl deacetylases. Recently, a COG2120 protein encoded in the chloroeremomycin gene cluster, Cep15, has been postulated to possess nucleotidyltransferase activity. Here, we demonstrate that Cep15 possesses no catalytic activity and does not have a clear role in chloroeremomycin biosynthesis. This result strongly suggests that cep15 and bal2 are evolutionary artifacts and may be pseudogenes. Comparative sequence analysis with the closely related active Orf2* deacetylase (teicoplanin biosynthesis) reveals an asparagine in place of a metal-binding histidine in the "pseudo-active site" of Cep15. Substitution of this histidine by asparagine in Orf2* abolishes deacetylase activity. Remarkably, the Cep15 N164H mutant is an active deacetylase. To our knowledge, this is the first example of reactivating an ancestral enzymatic role for a bacterial protein by point mutagenesis.