INTERDOMAIN BINDING OF NADPH IN P-HYDROXYBENZOATE HYDROXYLASE AS SUGGESTED BY KINETIC, CRYSTALLOGRAPHIC AND MODELING STUDIES OF HISTIDINE-162 AND ARGININE-269 VARIANTS

The conserved residues His-162 and Arg-269 of the flavoprotein p-hydro xybenzoate hydroxylase (EC 1.14.13.2) are located at the entrance of t he interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis, The catalytic properties of H162 R, H162Y, and R269K were similar to the wild-type enzyme. However, les s conservative His-162 and Arg-269 replacements strongly impaired NADP H binding without affecting the conformation of the flavin ring and th e efficiency of substrate hydroxylation, The crystal structures of H16 2R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 Angstrom resolution, respectively. Both structures are virtually i ndistinguishable from the wild-type enzyme-substrate complex except fo r the substituted side chains, In contrast to wild-type p-hydroxybenzo ate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate, NA DPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyroc arbonate inactivation, suggesting that His-162 is involved in NADPH bi nding, Based on these results and GRID calculations we propose that th e side chains of His-162 and Arg-269 interact with the pyrophosphate m oiety of NADPH, An interdomain binding mode for NADPH is proposed whic h takes a novel sequence motif (Eppink, M, H, M,, Schreuder, H, A., an d van Berkel, W, J, H, (1997) Protein Sci, 6, 2454-2458) into account.