In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity. Here we fill such gaps by analyzing anticorrelating occurrences of genes across species. Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins. So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway. For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity. The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity.

New protein function is thought to evolve mostly by gene duplication and divergence. Here we present phylogenetic evidence that the multifunctional protein Fis of the gamma proteobacterial species derived from the COOH-terminal domain of an ancestral alpha proteobacterial NtrC transcriptional regulatory protein. All of the known enterobacterial fis genes are preceded by an open reading frame, named yhdG, that is highly similar to nifR3, a gene that forms an operon with ntrC in several alpha proteobacterial species. Thus, we propose that yhdG and fis were acquired by a lineage ancestral to the gamma proteobacteria in a single horizontal gene transfer event, and later diverged to their present functions.