The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K i Ϸ 1 nM). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.Despite the accumulation of sequence information from a large diversity of species and phyla, newly sequenced bacterial genomes continue to reveal a high proportion of genes of unknown function (1), including a significant subset of "ORFans" (2), i.e. putative open reading frames (ORFs) 1 without significant similarity to any previously encountered protein (or conceptual translation) sequences. Most genes found in data bases have only been predicted by computer methods and never experimentally validated. It is thus expected that some annotated ORFs, in particular among the ORFans, might not correspond to real genes. In a previous study, we verified the existence of a cognate transcript for 25 Escherichia coli ORFans with a surprising rate of success (92%) (3). Given that most ORFans appear to be transcribed, we have now initiated a systematic expression and structure determination program for the proteins encoded by these (apparently) unique genes. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, it is expected that some ORFans should exhibit structural similarity to previously described protein families, hence providing some functional hints. Alternatively, targeting ORFans for structure determination is also a suitable strategy to optimize the discovery of original protein folds, one of the goals of structural genomics.In a pilot study involving five ORFan genes, we succeeded in producing four of them in E. coli as soluble proteins, and we report here the most advanced project, ykfE. YkfE (Swiss-Prot accession number P45552; b0220 in the Blattner data base (4)) is a 474-nucleotide-long uncharacterized ORF. It is part of a single gene operon and was found to exhibit a high level of expression during the exponential...