showing mutations installed during each round. b) Time course for the formation of N-hydroxycytidine 4 from cytidine 2 (50 mM) catalyzed by CAT3 (2.5 μM, red), CAT2 (2.5 µM, green), CAT1 (2.5 µM, orange), wild-type CD (2.5 μM, blue) and no enzyme (black) in the presence of 1% NH2OH, pH 7. The evolved CD variants show initial accumulation of N-hydroxy-cytidine 4 beyond the equilibrium position followed by slower redistribution of products to equilibrium. c) HPLC traces showing cytidine 2 (500 mM) conversion to N-hydroxy-cytidine 4 and uridine 3 catalyzed by CAT3 (25 µM, red) and wildtype CD (25 μM, blue) in the presence of 10% NH2OH, pH 7. Retention times of 3 and 4 are identical to authentic standards. d) The active site of CD with uridine bound (PDB code: 1AF2). Mutations installed in round 1 (T123G) are shown as orange spheres, round 2 (N72P A88G L149V) as green spheres, and round 3 (A53E T125S I147V) as red spheres. N125T from round 2 is not included as this was subsequently mutated in round 3. The uridine ligand is shown as atom-coloured sticks with black carbons and the Zn 2+ cofactor is shown in grey. Zn 2+ binding residues His102, Cys 129 and Cys132, and catalytic Glu104 are shown as atom-coloured sticks with blue carbons.