Directed evolution of angiotensin‐converting enzyme 2 peptidase activity profiles for therapeutic applications

Figures

• 

  FIGURE 1. Directed evolution of angiotensin‐converting enzyme 2 (ACE2) as a therapeutic agent. (a) Key ACE2 peptidase reactions relevant to the Ang‐II/ATR therapeutic axis. Green background denotes hydrolysis reaction with beneficial in vivo effects. Red background denotes hydrolysis reaction with detrimental effects. Yellow background indicates unknown and potential for detrimental and/or beneficial, that is, reduction in amount of Ang‐I hydrolytic conversion to Ang‐II by the ACE dipeptidase, effects. (b) LC chromatograms showing peptide hydrolysis by ACE2‐displaying yeast. Our ACE2 system shows high peptidase activity on Ang‐II and Apelin‐13 substrates, and undetectable hydrolysis of Ang‐I. We used horseradish peroxidase‐displaying yeast as a negative control. (c) The ACE2 substrate binding pocket is composed of two subsites: S1 that encases the substrate residue N‐terminal to the cleavage site and S1′ that accommodates the final C‐terminal residue. The labeled residues correspond to positions for which we constructed and screened yeast‐displayed NNB mutant libraries. The three circled residues indicate amino acid positions where mutations enhanced activity and/or specificity. (d)…

• 

  FIGURE 2. Kinetic properties of engineered ACE2 variants. (a) Initial rate plots for single substrate hydrolysis of Ang‐II, Apelin‐13, and Ang‐I peptides. Error bars denote standard deviations for duplicate measurements and the absence of error bars for some data points indicates a standard deviation less than height of data point marker. The lines depict nonlinear regression fits to the Michaelis–Menten equation and the kinetic parameters are reported in Table 1. (b) Initial rate plots zoomed in to lower substrate concentrations that are relevant for physiological functioning in vivo. (c) Relative amounts of Ang‐II and Apelin‐13 hydrolysis products formed in mixed peptide substrate assays. Ang‐I, Ang‐II, and Apelin‐13 were present at identical concentrations in each reaction and incubated with 250 pM purified ACE2. Activity values are normalized to one for wild‐type ACE2. Error bars denote standard deviations (SDs) for duplicate measurements. Error bars for wild‐type ACE2 values reflect SD in unnormalized hydrolysis product values for two trials. Error bars for mutant ACE2 values reflect SD in normalized hydrolysis product values. The slow rate of Ang‐I hydrolysis relative to Ang‐II and…

• 

  FIGURE 3. Peptide specificity profiles of engineered ACE2 variants. Purified ACE2s were incubated with 1 μM of peptide substrates in single substrate hydrolysis experiments. The engineered variants retain wild‐type‐like Ang‐II hydrolysis, while having substantially reduced activity on Apelin‐13 and dR9‐bk. Error bars denote standard deviation for duplicate measurements.