Figure 5

Indicators of gene function suggest changes in metallocarboxypeptidase paralog activity and specificity, yet purifying selection to maintain function. Predicted cDNA and amino acid sequences were curated for all duplicated members of the A/B subfamily of metallocarboxypeptidases found in Ensembl. (a, b) Predicted proteins were classified as active enzymes, pseudoenzymes (containing substitutions at active site residues), or pseudogenes (containing large deletions or other deleterious structural mutations). (c, d) Substrate specificity for each predicted protein was predicted based on the identity of the bovine CPA1 residue 255 equivalent. Hydrophobic = hydrophobic residue 255; acidic = basic residue 255; basic = acidic residue 255; polar = polar residue 255. (e, f) The predicted coding sequences for each paralog pair were used in a codon-based test of purifying selection, where greater Ds–dN indicates greater probability of purifying selection. The variance of the difference of synonymous and nonsynonymous substitutions per site was computed using the analytical method. Analyses were conducted using the Nei–Gojobori method in MEGA7. (g) The probability of rejecting the null hypothesis of strict-neutrality (dN = dS) in favor of the alternative hypothesis (dN < dS, purifying selection) is shown. A t-test was used to compare CPO paralogs with all others.