Fig. 3

Screening and characterization of superior mutants. Residues selected for steric hindrance mutagenesis in wild-type BbPheDH (a), and mutant M1–3 (b), M2–2 (c), and M3–2 (d). Screening of superior mutants (e) and kinetic parameters of BbPheDH and superior mutants (f–h). Residues shown in gray bubbles, including in blue bubbles (a–d), were all close (within 6 Å) to the side-chain phenyl ring of 2-OPBA, while the residues in blue bubbles (b–d) are not only close to the side-chain phenyl ring of 2-OPBA, but also close to the focal residues (G309, V306, and G144, respectively). Activity was measured in NH₄Cl/NH₄OH buffer (2 M, pH 9.5) containing 1–20 mM 2-OPBA and 0.5 mM NADH at 30 °C and carried out at a 200-μL scale in 96-well microtiter plates by monitoring the initial decrease velocity of the absorbance at 340 nm (indicating NADH consumption). All determinations were performed in triplicate, and error bounds represent ± sd