Reversing a point mutation... produced a functional enzyme.

Publishing in Chemistry & Biology Truman et al. have identified a single amino acid substitution in the active site of the protein Cep15, which is part of the glycopeptide antibiotic chloroeremomycin biosynthetic machinery in Amycolatopsis orientalis, that abolishes its catalytic activity. Reversing a point mutation in the cep15 gene produced a functional enzyme. This finding represents the first time that the function of a bacterial pseudogene has been restored by a point mutation.

Glycopeptides, including teicoplanin and vancomycin, are used to treat patients with multidrug-resistant bacterial infections. Teicoplanin has a glucosaminyl group that results from glycosyl transfer and modification by a COG2120 domain deacetylase. Other glycopeptides, such as chloroeremomycin and balhimycin, lack a glucosaminyl group, although the gene clusters that code for these antibiotics still contain a COG2120 domain deacetylase gene. Truman and colleagues focused on the COG2120 domain protein of the chloroeremomycin cluster, Cep15, to pin down its catalytic function.

Previous reports had assigned either a nucleotidyltransferase function or an N-acetylglucosaminyl deacetylase function to Cep15, but Truman et al. found that purified Cep15 has neither of these functions and proposed that cep15 is a non-functional pseudogene. The cep15 homologue in the teicoplanin cluster was shown to encode a functional deacetylase, whereas the cep15 homologue in the balhimycin cluster was a pseudogene. Inspection of sequence alignments revealed that an active-site histidine (His164) had been replaced with an asparagine in the non-functional deacetylases. On the basis of crystal structures of related eukaryotic COG2120 proteins, the same histidine was identified as a ligand for zinc, a cofactor that is essential for catalytic activity. Mutating the cep15 Asn164 to His164 restored deacetylase activity. This confirmed that the active-site His164 has a pivotal role in the function of these COG2120 enzymes. Pseudogenes are usually removed from streamlined bacterial genomes, so the authors speculate that cep15 might have an as-yet-unidentified role.

Reactivation of minimally deactivated genes could provide a new source of enzymatic activities for antibiotic engineering.