Biochemical Characterization of Yeast Aad Proteins

Heterologous expression, purification, and biochemical characterization of yeast Aad proteins were carried out in parallel, with the PcAad1p serving as a reference. PcAad1p and all recombinant yeast Aad proteins bear an N-terminal His₆-GST and a C-terminal His₆ tag and were purified following the GST-affinity batch purification (10 (link)). The activities of purified reference PcAad1p, yeast Aadp proteins, and mutated yeast Aad recombinant proteins were assayed against a panel of aliphatic/aromatic aldehydes and aryl-alcohols using both NAD(P)H and NAD(P)⁺ as reduction and oxidation cofactors (10 (link)). Reactions were quantified spectrophotometrically by following the consumption or production of cofactor NAD(P)⁺(H) at λ = 340 nm (ε₃₄₀ = 6.2 mM⁻¹ · cm⁻¹) in 250-μl microplates containing 0.3 mM cofactor and 0.3 mM substrates (10 (link)) (microplate reader model 680XR; Bio-Rad). Kinetic parameters (K_m and V_max) quantifying NADP⁺(H) consumption/formation were assayed at 355 nm (ε₃₅₅ = 5.12 mM⁻¹ · cm⁻¹) using a UV-visible spectrophotometer (Shimadzu UV1800) in 1-ml cuvettes, as described previously; the substrate absorption at this wavelength is negligible (10 (link)). For each recombinant protein, significant differences relative to a pGS-21a blank plasmid were calculated using a 2-tailed t test at a P value of <0.05.