Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding.
Arg44, located at the si-face side of the flavin ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180 degrees in 50% of the molecules. The phi, psi angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.
Eppink
M H
MH
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
Schreuder
H A
HA
Van Berkel
W J
WJ
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D008239:Lysine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D009249:NADP; D001120:Arginine; D015109:4-Hydroxybenzoate-3-Monooxygenase; D008239:Lysine