1BKW

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

Summary for 1BKW

• Entry DOI: 10.2210/pdb1bkw/pdb
• Descriptor: PROTEIN (P-HYDROXYBENZOATE HYDROXYLASE), FLAVIN-ADENINE DINUCLEOTIDE, P-HYDROXYBENZOIC ACID, ... (4 entities in total)
• Functional Keywords: hydroxybenzoate, oxidoreductase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 45260.16

Authors

Eppink, M.H.,Schreuder, H.A.,Van Berkel, W.J. (deposition date: 1998-07-13, release date: 1998-07-22, Last modification date: 2023-08-09)

Primary citation

Eppink, M.H.,Schreuder, H.A.,Van Berkel, W.J.
Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding.
Eur.J.Biochem., 231:157-165, 1995

PubMed Abstract: 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.

PubMed: 7628466
DOI: 10.1111/j.1432-1033.1995.0157f.x

Experimental method

X-RAY DIFFRACTION (2.2 Å)