PDBsum entry 2phh

Oxidoreductase

PDB id: 2phh

Contents

Protein chain

391 a.a. *

Ligands

APR

PHB

Waters ×57

* Residue conservation analysis

PDB id:

2phh

Name:

Oxidoreductase

Title:

The coenzyme analogue adenosine 5-diphosphoribose displaces fad in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation

Structure:

P-hydroxybenzoate hydroxylase. Chain: a. Engineered: yes

Source:

Pseudomonas fluorescens. Organism_taxid: 294

Biol. unit:

Dimer (from PQS)

Resolution:

2.70Å R-factor: 0.168

Authors:

J.M.Van Derlaan,J.Drenth,W.G.J.Hol

Key ref:

J.M.van der Laan et al. (1989). The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation. Biochemistry, 28, 7199-7205. PubMed id: 2819062 DOI: 10.1021/bi00444a011

Date:

19-Jun-89 Release date: 15-Oct-90

Protein chain

P00438  (PHHY_PSEFL) -  p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

Seq: 394 a.a.

Struc: 391 a.a.

Enzyme reactions

• Enzyme class: E.C.1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase.
• Pathway: Benzoate Metabolism
• Reaction: 4-hydroxybenzoate + NADPH + O2 + H⁺ = 3,4-dihydroxybenzoate + NADP⁺ + H2O

4-hydroxybenzoate (Bound ligand (Het Group name = PHB) corresponds exactly) + NADPH + O2 (Bound ligand (Het Group name = APR) matches with 71.43% similarity) + H(+) = 3,4-dihydroxybenzoate + NADP(+) + H2O

• Cofactor: FAD

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1021/bi00444a011

Biochemistry 28:7199-7205 (1989)

PubMed id: 2819062

The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.

J.M.van der Laan, H.A.Schreuder, M.B.Swarte, R.K.Wierenga, K.H.Kalk, W.G.Hol, J.Drenth.

ABSTRACT

p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.