1bgj

P-HYDROXYBENZOATE HYDROXYLASE (PHBH) MUTANT WITH CYS 116 REPLACED BY SER (C116S) AND HIS 162 REPLACED BY ARG (H162R), IN COMPLEX WITH FAD AND 4-HYDROXYBENZOIC ACID

spinqualitylabelsall models 1bgj, resolution 3.00Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 1bgj is a 1 chain structure with sequence from Pseudomonas fluorescens. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 3Å Ligands: FAD, PHB Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function PHHY_PSEFL Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed 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. Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants.,Eppink MH, Schreuder HA, van Berkel WJ J Biol Chem. 1998 Aug 14;273(33):21031-9. PMID:9694855 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Hydroxylases 3D structures

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