1BGN

P-HYDROXYBENZOATE HYDROXYLASE (PHBH) MUTANT WITH CYS 116 REPLACED BY SER (C116S) AND ARG 269 REPLACED BY THR (R269T), IN COMPLEX WITH FAD AND 4-HYDROXYBENZOIC ACID

Summary for 1BGN

• Entry DOI: 10.2210/pdb1bgn/pdb
• Descriptor: P-HYDROXYBENZOATE HYDROXYLASE, FLAVIN-ADENINE DINUCLEOTIDE, P-HYDROXYBENZOIC ACID, ... (4 entities in total)
• Functional Keywords: oxidoreductase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 45232.08

Authors

Eppink, M.H.M.,Schreuder, H.A.,Van Berkel, W.J.H. (deposition date: 1998-05-29, release date: 1998-08-12, Last modification date: 2024-05-22)

Primary citation

Eppink, M.H.,Schreuder, H.A.,van Berkel, W.J.
Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants.
J.Biol.Chem., 273:21031-21039, 1998

PubMed Abstract: The conserved residues His-162 and Arg-269 of the flavoprotein p-hydroxybenzoate hydroxylase (EC 1.14.13.2) are located at the entrance of the interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis. The catalytic properties of H162R, H162Y, and R269K were similar to the wild-type enzyme. However, less conservative His-162 and Arg-269 replacements strongly impaired NADPH binding without affecting the conformation of the flavin ring and the efficiency of substrate hydroxylation. The crystal structures of H162R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 A resolution, respectively. Both structures are virtually indistinguishable from the wild-type enzyme-substrate complex except for the substituted side chains. In contrast to wild-type p-hydroxybenzoate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate. NADPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyrocarbonate inactivation, suggesting that His-162 is involved in NADPH binding. Based on these results and GRID calculations we propose that the side chains of His-162 and Arg-269 interact with the pyrophosphate moiety of NADPH. An interdomain binding mode for NADPH is proposed which takes a novel sequence motif (Eppink, M. H. M., Schreuder, H. A., and van Berkel, W. J. H. (1997) Protein Sci. 6, 2454-2458) into account.

PubMed: 9694855
DOI: 10.1074/jbc.273.33.21031

Experimental method

X-RAY DIFFRACTION (2 Å)