After a decade of comprehensive genomic sequencing, more than 500 genomes have been sequenced to completion, mostly prokaryotes. The prodigious rate of new sequence annotation is highlighted by the fact that there were just over 300 genomes available when this study was carried out in late 2004. However, the fraction of genes for which no function can be predicted remains high (30%–50%). In response, proposals have been put forth for the bioinformatics analysis of bacterial genomes to identify genes with high likelihood of scoring true in confirmatory laboratory assays of their respective function 12. This would increase the field's pool of experimentally characterized proteins, with concomitant increases in the accuracy and coverage of genome annotation. We believe the return on investment of this approach would be particularly high when addressing the problem of orphan activities, that is, enzymatic activities for which no sequence information is available 34.

Decades of detailed enzymology have created a wealth of knowledge about enzymes and their activities. However, crucial aspects of these enzymes are absent from bioinformatics databases with surprising frequency. For example, recent computational analyses of sequence databases demonstrate that at least 36% of enzyme activities that have been assigned an Enzyme Commission (EC) number 5 appear to be devoid of a gene or protein sequence 3. Since then, similar analyses have been published, with similar results 467. The existence of such a large fraction of orphan activities is surprising, given that many of these enzymes have been described decades ago and are often involved in basic cellular functions. Several examples exist of the recent identification of genes involved in important enzymatic functions (reviewed in 12). Indeed, in our study 44 orphans were found to be present in one or more primary metabolic pathways in a variety of species (described below). Details of many of the orphan enzymes uncovered during this survey point to multiple and significant consequences for the lack of sequence information in areas such as genome annotation, computational pathway prediction, and metabolic engineering. For these reasons, the orphan problem and related issues were highlighted in a recent report of the American Society for Microbiology 8. In view of the biological richness associated with orphan enzymatic activities (Figure 1, Table 1), we have taken the first steps in creating the foundations of an Enzyme Genomics Initiative 3.

Here we describe a literature-based survey of presumed orphans intended to further validate and characterize these activities (Figure 2). The confidence of the results of this survey was designed to be within a 5% error margin relative to the universe of orphan activities, based on a randomly selected subset of orphan activities from the Nomenclature Committee of the IUBMB (NC-IUBMB). We have also assessed the practicability of identifying the genes associated with these orphans. As a consequence, the survey captures data from the literature that should facilitate the identification of cognate genes for the orphan activities evaluated. Here, we define the cognate gene for an activity as a gene that has been shown to code for an enzyme that carries out that activity.

The survey confirmed that ~80% of the sampled orphans do not have sequence information associated with them. Consequently, this lack represents a true information deficit. Weaknesses in database integration and a lack of information capture from the literature to databases appear to be largely responsible for most of the artifactual orphans making up the other 20%. Given the importance of these enzymatic activities, we propose that the public sequence databases assign high priority to correcting database entries for artifactual orphans. We further propose that a systematic effort be undertaken to sequence the genes of validated orphans, as this survey demonstrates that primary literature data and database analyses combined with current proteomics and genomic technologies should be adequate to enable the rapid identification of many of these genes.

Most orphan enzymatic activities are bona fide (Table 2). Our survey found that more than 80% of orphans are not due to artifacts such as missing database annotations (primarily failure to capture information from the literature), or lack of database cross-referencing, such as the availability of a sequence in one database not being reflected in a second database. Specifically, a total of 187 orphans out of 228 surveyed activities were validated in at least one of 287 species (species are listed in Table 3 and Table 4, the list of validated orphans is in Table 5). A majority of orphans (54.36%) occurred in Eukaryotes, followed by Eubacteria (39.37%) (Table 6). Within the Eubacteria, genus Pseudomonas was significantly overrepresented (35%) (Table 7). While a systematic determination of the species spectrum of orphan activities was not performed here, we did notice several cases of an orphan activity reported in more than one species, as well as one case of an orphan activity occurring in species from different domains.

Because the eventual isolation of the cognate genes of these activities is greatly facilitated by comprehensive genome sequencing, we determined for what fraction of all validated orphans a full genome sequence is available (Table 8). 43% of Eubacterial species in which orphans occurred were found to have such sequences, available either presently or due shortly. This figure rises to 83% when including the genomes of related species, on the assumption that they might be sufficiently closely related to permit the identification of the cognate gene. For example, at the time of this study the completed genome sequence of Pseudomonas fluorescens was not available, but those of three other Pseudomonas species were.

Oxidoreductases (EC1) and transferases (EC2) were the most frequently represented classes of enzymatic activity for validated orphans (Figure 3). On a per capita basis, oxidoreductases and transferases were overrepresented by ~20%, whereas hydrolases and ligases were underrepresented by 35% and 64%, respectively.

The original publication date for all orphans was broadly distributed around a mean of 1977 (Figure 4), compared to a mean of 1975 for validated orphans.

This survey demonstrates that ~80% of orphan enzymatic activities are bona fide; therefore, we conclude that of the 1,356 putative orphans extant at the time of this study, more than 1,000 are highly likely to constitute true information deficits since their lack of sequence information is not the result of a database error.

The absence of DNA or protein sequences encoding such well-characterized enzymatic activities is particularly consequential because these activities were often identified decades ago, and many have been the focus of significant research activity (Table 1). Without the cognate sequences for these activities, the quality of annotation of all sequenced genomes in terms of both coverage (fraction of genes that can be recognized) and accuracy (fraction of predicted gene functions that are correct) is diminished. Many of these activities may go for years without being sequenced – for example, 1-acylglycerophosphocholine O-acyltransferase (Table 1) was finally purified and sequenced nearly forty years after it was first characterized 10. Perhaps more troubling is the unknown pool of "false positive" annotations. Phosphogluconate 2-dehydrogenase (Table 1), an orphan at the time of this analysis, has since been assigned to a sequence in the human genome with no experimental evidence linking it to that or any homologous sequence, but apparently instead on the basis of the gene in question already being assigned a similar activity. This kind of "hidden orphan" would have been missed by most orphan analyses, and can be expected to propagate a potentially incorrect assignment to other genomes in the future. Computational metabolic pathway prediction 11 and metabolic engineering also depend on sequence information and are thus similarly compromised.

Conversely, ~20% of orphans surveyed were observed to be artifacts, such that ~270 orphans out of 1,356 putative orphans examined should be resolvable entirely via literature research and database cleanup. As a result of this process as it was carried out on our sampling of orphans, we have reported 11 artifactual orphan activities to public sequence repositories for correction (see Table 8 for examples).

In addition to validating orphans, the survey was useful in capturing information from the literature to assess their salvageability: more than half of validated orphans were found to be salvageable (Figure 5). Examples of salvageable orphan activities with the traits that make them salvageable are listed in Table 11.

As abundantly noted elsewhere, such database cleansing is essential to maximize the existing research investment and prevent the propagation of mistakes 121314 (see Table 12 for examples of artifacts that have been resolved). This necessity has not eluded the field of enzymology 341516, and the present survey demonstrates the usefulness of correlating biological databases and mining the literature to enhance the value of existing research and facilitate the identification of the remaining orphan-associated genes. Until recently, there were no general repositories of orphan activity data, although some species-specific databases and pages were maintained, such as EchoBase 17 and a web page listing unidentified E. coli enzymes maintained by the EcoCyc project 18. Consequently, we updated the MetaCyc 19 database to identify reactions that have been analyzed by this survey, and annotated them and associated database objects with results such as the validity of their orphan status, links to their cognate protein in the case of artifacts, and the properties of the protein copurifying with the activity in the case of validated orphans. Recently, Lespinet and Labedan created ORENZA 20, a database dedicated to maintaining an up-to-date listing of all enzyme activities for which no sequences are available in major sequence databases 6. We are contributing our updated orphan information to ORENZA as well. These data, captured in MetaCyc and ORENZA, should facilitate the work of enzymologists interested in identifying the cognate genes of orphan activities. For instance, the work of Melnick et al. 21 is an excellent example of the combined application of modern laboratory and bioinformatics techniques that would benefit from the data described here.

Several proposals have been made recently aimed at producing a complete catalog of biochemical activities, biological functions, and their cognate genes 23. Many of these proposals recommend that such a project begin with prokaryotes because of the general ease of gene cloning from these species 12. Indeed, our data support this notion, as we find substantially more orphans with a salvageability ranking of "good" and "excellent" in prokaryotes as compared to eukaryotes. The availability of a comprehensive review of the problem achieved by this survey, combined with broad genomic sequencing and powerful computational tools, leads us to conclude that the field is in an excellent position to rectify the information gap associated with the orphan activity phenomenon.

More than one third of enzyme activities with assigned EC numbers are orphan activities, having no associated gene or protein sequence. We carried out a literature-based survey of a representative sample of presumed orphans intended to further validate and characterize these orphan activities. We have also assessed the practicability of identifying the genes associated with these orphans. In doing so, we captured data from the literature that should assist in future identification of cognate genes for the orphan activities we examined.

This survey confirmed that about 80% of sampled orphan activities have no sequence information associated with them, either in databases or in the literature. Weaknesses in database integration and failure to capture information from the literature account for most of the remaining 20%.

This survey points toward the significant scientific cost of having such a large fraction of characterized enzyme activities disconnected from sequence data. It also suggests that a larger effort, beginning with a comprehensive survey of all putative orphan activities, would resolve nearly 300 artifactual orphans and reconnect a wealth of enzyme research with modern genomics. For these reasons, we propose that a systematic effort to identify the cognate genes of orphan enzymes be undertaken.

Enzyme Commission (EC), Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), Structured Query Language (SQL)

PK conceived the study. PK and YP jointly devised the methodology. YP performed the literature research and drafted the manuscript. PK revised the manuscript.