Surface loops may provide additional function necessary for enzyme-to-pseudoenzyme transition in the fungal...

The transition from enzyme to pseudoenzyme is thought to begin with moonlighting enzymes that have gained nonenzymatic functions. Subsequent gene duplication events allow for the separation of enzyme and pseudoenzyme function. We explored this enzyme-to-pseudoenzyme transition in the family of fungal metallocarboxypeptidases through bioinformatics approaches, molecular modeling, and biochemical analyses. Over 3000 predicted fungal metallocarboxypeptidases were first classified by phylogeny and active site signature into 14 clusters. Prediction of isoelectric point revealed potential subcellular location, while predictions of solvent accessible surface area and AlphaFold modeling of representative structures suggested a tendency for clusters rich in pseudoenzymes to have extensive surface loops and polar distribution of surface electrostatic potential, possible requirements for the addition of nonenzymatic function. Five basidiomycete carboxypeptidases were selected for experimental analysis by RNA-seq, western blotting following expression in Sf9 and HEK293T systems, and enzymatic activity. No activity was detected from predicted pseudoenzymes, either purified or unpurified. Both predicted-active enzymes were secreted from Sf9 cells, although only one could be purified, with expected carboxypeptidase activity and specificity toward large hydrophobic C-terminal amino acids. Altogether, our study suggests that the addition of surface loops may be a key feature in the acquisition of pseudoenzyme function, and that both enzymes and pseudoenzymes are likely to play important and unique roles in these fungal systems.