1bkw | pdb_00001bkw
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p-Hydroxybenzoate hydroxylase (phbh) mutant with cys116 replaced by ser (c116s) and arg44 replaced by lys (r44k), in complex with fad and 4-hydroxybenzoic acid
Overview
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.
About this Structure
1BKW is a Single protein structure of sequence from Pseudomonas fluorescens with FAD and PHB as ligands. Active as 4-hydroxybenzoate 3-monooxygenase, with EC number 1.14.13.2 Full crystallographic information is available from OCA.
Reference
Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding., Eppink MH, Schreuder HA, Van Berkel WJ, Eur J Biochem. 1995 Jul 1;231(1):157-65. PMID:7628466
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