Figure 7

Xenopus tropicalis CPO orthologs exhibit different substrate preferences. (a) One hundred microliters of each trypsinized media was incubated with 900 µl of 0.5 mM substrate (FA-EE, FA-FA, FA-FF, pH 7.5) at room temperature. Change in absorbance at 340 nm was measured over time and the rate of reaction shown as the change in absorbance (milli-absorbance-units) per minute. n = 3–6. Error bars indicate standard deviation. *p < 0.05, comparing to the corresponding WTV dataset, as determined by ANOVA and Tukey–Kramer post-hoc analysis. (b) Each Xenopus tropicalis CPO paralog was modeled with AlphaFold2 and aligned in Pymol with X-ray crystal structures for Bos taurus CPA (3CPA) and Homo sapiens CPO (5MRV, chain a). All images show the zinc cofactor from Hs CPO as a gray sphere, the zinc cofactor from Bt CPA as a yellow sphere (largely superimposed by the gray sphere) and the Gly-Tyr dipeptide bound to the active site of Bt CPA as a yellow stick model. Key active site residues from each structure are indicated. Cavity surfaces, as viewed from the inside of the protein and with the prodomains removed, are shown in dark grays, while the protein outer surface is shown with white carbons, red oxygens, and blue nitrogens. No substrate binding pocket is shown for Bt CPA, as the pocket is filled with the Gly-Tyr and so not rendered as a surface.