Revealing the hidden functional diversity of an enzyme family

Abstract

Millions of protein database entries are not assigned reliable functions, preventing the full understanding of chemical diversity in living organisms. Here, we describe an integrated strategy for the discovery of various enzymatic activities catalyzed within protein families of unknown or little known function. This approach relies on the definition of a generic reaction conserved within the family, high-throughput enzymatic screening on representatives, structural and modeling investigations and analysis of genomic and metabolic context. As a proof of principle, we investigated the DUF849 Pfam family and unearthed 14 potential new enzymatic activities, leading to the designation of these proteins as β-keto acid cleavage enzymes. We propose an in vivo role for four enzymatic activities and suggest key residues for guiding further functional annotation. Our results show that the functional diversity within a family may be largely underestimated. The extension of this strategy to other families will improve our knowledge of the enzymatic landscape.

Panoramic view of a superfamily of phosphatases through substrate profiling

“…Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3,4). Family-wide substrate profiling offers a data-rich resource.…”

Enzyme Promiscuity: Engine of Evolutionary Innovation

“…Orthogonal to the genome-wide study discussed above, Bastard et al (30) demonstrated the use of an integrated strategy for exploring the functional diversity of a previously undescribed enzyme family, DUF849 (see Fig. 1 for general reaction and representative substrates).…”

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

“…Many of the widespread DUFs in bacteria are biologically essential, and the importance of prioritizing these DUFs has been recognized in recent years (10). Before this study, only one DUF family, the DUF849 family of 922 proteins, had been evaluated in large scale by a single report (11), which heavily relied on previously published enzymatic activity and catalytic mechanism/ liganded structure of one of its members (35,36). Recognizing the difficulties associated with DUF functional assignment, we attempt to apply an integrated "genomic enzymology" strategy that had only been used for functional characterization of members of families with known functions (37,38).…”

“…However, the assignment of functions to uncharacterized proteins is challenging (11)(12)(13). The methods now available for discovering the functions of uncharacterized proteins, including DUFs, are inefficient and often depend on inference from the functions of characterized homologs (11). Therefore, new strategies are required to confront and solve the functional assignment challenge.…”

“…Thus, reliable experimental identification of the in vitro enzymatic activities and in vivo physiological functions for the DUF families is important. However, the assignment of functions to uncharacterized proteins is challenging (11)(12)(13). The methods now available for discovering the functions of uncharacterized proteins, including DUFs, are inefficient and often depend on inference from the functions of characterized homologs (11).…”