Evolution of enzymatic function through gene duplication in the metallocarboxypeptidase family

Changes in the genetic code occur frequently through, for example, DNA breaks and recombination events, often leading to the duplication of genes. The M14 family of metallocarboxypeptidases suggests such a history of gene duplication. In order to understand the functional outcome of such events, we investigated more recent duplication events in the M14 family of enzymes through analysis of genomic data found in the Ensembl genome database. Of the 23 M14 genes found in most vertebrates, four genes were present as two or more copies in many fish species. The AEBP1, CPXM1, and CPZ genes were duplicated in most fish with an arrangement suggesting a large-scale event such as whole-genome duplication. The CPO gene was also frequently duplicated in many fish species, but with copies tandemly arranged, suggestive of crossing-over errors. A close analysis of the synteny of four CPO paralogs found in Xenopus tropicalis suggested potential transposon-mediated duplication. Prediction of enzyme functions for the products of these genes showed that a greater amount of neofunctionalization was present in CPO paralogs as compared with other carboxypeptidase enzymes; purifying selection to maintain function remained detectable. To further examine function, the four CPO paralogs found in Xenopus tropicalis were examined biochemically. All were expressed in HEK293T cells, but no enzymatic activity was detected. Subcellular distribution suggested a possible function on lipid droplets similar to that previously shown for human CPO. Experiments to further examine enzyme activity using the Sf9 expression system are ongoing. © FASEB.