Fig. 5: The identification of ADO residues involved in substrate binding and turnover. | Nature Communications

Fig. 5: The identification of ADO residues involved in substrate binding and turnover.

From: An mRNA-display derived cyclic peptide scaffold reveals the substrate binding interactions of an N-terminal cysteine oxidase

Fig. 5

a A sequence alignment comparing the amino acid composition of different NCOs, including ADOs (human, mouse and fruit fly) and PCOs (arabidopsis, rice and wheat). Red background and white text highlights conserved residues. M denotes residues involved in metal cofactor coordination. An asterisk (*) denotes residues mutated during this study. A black asterisk denotes residues mutated for substrate binding and activity measurements. A red asterisk (*) denotes ADO-E92, which was mutated for CP6 characterisation. b The active site of cobalt-incorporated ADO (dark pink) in complex with CP-L8K-Ser (light blue), highlighting the residues mutated in this study (opaque). c Biophysical and activity analysis of ADO mutants with RGS52-15 (black) or IL322-15 (grey). (Top panel) The fold difference in equilibrium dissociation constant (KD) relative to wild type ADO (KD calculated as the geometric mean of a minimum of three independent SPR measurements (n = 3); error bars show the standard error). D206A (bold italics) displayed no binding. (Bottom) The specific activity relative to wild type ADO. Conducted at 37 °C under aerobic conditions using 1 mM substrate. The average of three independent experiments (n = 3) is shown (error bars show the standard error). Source data are provided as Source Data file.

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