PDBsum entry 1pbb

Oxidoreductase

PDB id: 1pbb

Contents

Protein chain

391 a.a. *

Ligands

FAD

DOB

Waters ×203

* Residue conservation analysis

PDB id:

1pbb

Name:

Oxidoreductase

Title:

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate, 2,4-dihydroxybenzoate and 2-hydroxy- 4-aminobenzoate and of the try222ala mutant, complexed with 2- hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring

Structure:

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

Source:

Pseudomonas fluorescens. Organism_taxid: 294

Biol. unit:

Dimer (from PQS)

Resolution:

2.50Å R-factor: 0.158

Authors:

H.A.Schreuder,A.Mattevi,W.G.J.Hol

Key ref:

H.A.Schreuder et al. (1994). Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring. Biochemistry, 33, 10161-10170. PubMed id: 7520279 DOI: 10.1021/bi00199a044

Date:

06-Jul-94 Release date: 30-Sep-94

Protein chain

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

Seq: 394 a.a.

Struc: 391 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

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 = DOB) matches with 90.91% similarity) + NADPH + O2 + H(+) = 3,4-dihydroxybenzoate + NADP(+) + H2O

• Cofactor: FAD

FAD (Bound ligand (Het Group name = FAD) corresponds exactly)

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

reference

DOI no: 10.1021/bi00199a044

Biochemistry 33:10161-10170 (1994)

PubMed id: 7520279

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.

H.A.Schreuder, A.Mattevi, G.Obmolova, K.H.Kalk, W.G.Hol, F.J.van der Bolt, W.J.van Berkel.

ABSTRACT

The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.