IDENTIFICATION OF A NOVEL CONSERVED SEQUENCE MOTIF IN FLAVOPROTEIN HYDROXYLASES WITH A PUTATIVE DUAL FUNCTION IN FAD NAD(P)H BINDING/

A novel conserved sequence motif has been located among the flavoprote in hydroxylases. Based on the crystal structure and site-directed muta genesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomon as fluorescens, this amino acid fingerprint sequence is proposed to pl ay a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-termin al part of helix H7. The conserved amino acids Asp 159, Gly 160, and A rg 166 are necessary for maintaining the structure. The backbone oxyge n of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact in directly with the pyrophosphate moiety of FAD, whereas it is known fro m mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.