Phe(161) and Arg(166) variants of p-hydroxybenzoate hydroxylase - Implications for NADPH recognition and structural stability

Phe(161) and Arg(166) of p-hydroxybenzoate hydroxylase from Pseudomonas flu orescens belong to a newly discovered sequence motif in flavoprotein hydrox ylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe(161) and Arg(166) were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent e nzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impa ired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 Angstrom is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 Angstrom revealed that Arg(166) is important for providing an intimate contact between the F AD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH. (C) 1999 Federat ion of European Biochemical Societies.