While the yeast Saccharomyces cerevisiae is a well-studied organism with its genome fully sequenced two decades ago, there remain several hundred uncharacterized open reading frames, ORFans, with no significant sequence homology to other genes. As ORFan roles cannot be determined solely through sequence homology, their functions remain a mystery until both in vitroand in vivobiochemical and cell biology experiments are performed. In collaboration with the Yeast ORFan Gene Project, we used bioinformatics modules to initially characterize one such ORFan, YIL165C.We found that together with the gene immediately preceding it, YIL164C (NIT1), the two genes have significant homology to the nitrilase superfamily of proteins but have low sequence homology to the other two S. cerevisiae nitrilases NIT2 and NIT3. Interestingly, the YIL164C protein alone contains both the conserved catalytic triad and a region that shares homology with prokaryotic nitrilases that specifically target aromatic, rather than aliphatic, nitrile substrates. We additionally experimentally verified that lab strains of S. cerevisiae, such as BY4741, SEY6210, and GA74, have a bonafide stop codon breaking up the two proteins in clones from both genomic DNA and mRNA, while the brewing yeast strain WLP001 (White Labs) and other related yeast strains have a single nucleotide polymorphism (SNP; A to G transition) allowing these two proteins to be expressed as a single, continuous polypeptide chain. To understand the function of both the full-length YIL164C/YIL165C and the truncated YIL164C protein, we expressed and purified the proteins in bacteria and began initial studies to understand their substrate preference. We hope to identify distinctions in activity, or the lack thereof, between the longer and the truncated proteins. Understanding these enzymes in vitro will provide clues to their in vivo functions and the evolutionary relationships among genes in this superfamily.

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Journal Article

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Koyama E, Walsh S

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