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

Citation:
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.
PDB ID: 1BGNDownload
MMDB ID: 55536
PDB Deposition Date: 1998/5/29
Updated in MMDB: 2017/12
Experimental Method:
x-ray diffraction
Resolution: 2  Å
Source Organism:
Similar Structures:
Biological Unit for 1BGN: dimeric; determined by author and by software (PISA,PQS)
Molecular Components in 1BGN
Label Count Molecule
Proteins (2 molecules)
2
P-hydroxybenzoate Hydroxylase
Molecule annotation
Chemicals (4 molecules)
1
2
2
2
* Click molecule labels to explore molecular sequence information.

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