Identifying pseudoenzymes using functional annotation: pitfalls of common practice

Pseudoenzymes are proteins that are evolutionary related to enzymes but lack relevant catalytic activity. They are usually evolved from enzymatic ancestors that have lost their catalytic activities. The loss of catalytic function is one extreme amongst the other evolutionary changes that can occur to enzymes, like the changing of substrate specificity or the reaction catalysed. However, the loss of catalytic function events remain poorly characterised, except for some notable examples, like the pseudokinases. In this review, we aim to analyse current knowledge related to pseudoenzymes across a large number of enzymes families. This aims to be a review of the data available in biological databases, rather than a more traditional literature review. In particular, we use UniProtKB as the source for functional annotation and M-CSA (Mechanism and Catalytic Site Atlas) for information on the catalytic residues of enzymes. We show that explicit annotation of lack of activity is not exhaustive in UniProtKB and that a protocol using lack of catalytic annotation as an indication for lack of function can be an adequate alternative, after some corrections. After identifying pseudoenzymes related to enzymes in M-CSA, we were able to comment on their prevalence across enzyme families, and on the correlation between lack of catalytic function and the mutation of catalytic residues. These analyses challenge two common ideas in the emerging literature: that pseudoenzymes are ubiquitous across enzyme families and that mutations in the catalytic residues of enzyme homologues are always a good indication of lack of activity.