PDBsum entry 2yyi

Oxidoreductase

PDB id: 2yyi

Contents

Protein chain

474 a.a. *

Ligands

SO4 ×4

FAD

Waters ×512

* Residue conservation analysis

PDB id:

2yyi

Name:

Oxidoreductase

Title:

Crystal structure of the oxygenase component (hpab) of 4- hydroxyphenylacetate 3-monooxygenase complexed with fad

Structure:

4-hydroxyphenylacetate-3-hydroxylase. Chain: a. Synonym: 4-hydroxyphenylacetate 3-monooxygenase. Engineered: yes

Source:

Thermus thermophilus. Organism_taxid: 300852. Strain: hb8. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.66Å R-factor: 0.169 R-free: 0.182

Authors:

S.-H.Kim,T.Hisano,K.Takeda,W.Iwasaki,A.Ebihara,K.Miki

Key ref:

S.H.Kim et al. (2007). Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8. J Biol Chem, 282, 33107-33117. PubMed id: 17804419 DOI: 10.1074/jbc.M703440200

Date:

30-Apr-07 Release date: 04-Sep-07

Protein chain

Q5SJP8  (HPAB_THET8) -  4-hydroxyphenylacetate 3-monooxygenase oxygenase component from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 481 a.a.

Struc: 474 a.a.

Enzyme reactions

• Enzyme class: E.C.1.14.14.9 - 4-hydroxyphenylacetate 3-monooxygenase.
• Reaction: 4-hydroxyphenylacetate + FADH2 + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O + H⁺

4-hydroxyphenylacetate + FADH2 (Bound ligand (Het Group name = FAD) corresponds exactly) + O2 = 3,4-dihydroxyphenylacetate + FAD + H2O + H(+)

• Cofactor: FAD

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

reference

DOI no: 10.1074/jbc.M703440200

J Biol Chem 282:33107-33117 (2007)

PubMed id: 17804419

Crystal structure of the oxygenase component (HpaB) of the 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8.

S.H.Kim, T.Hisano, K.Takeda, W.Iwasaki, A.Ebihara, K.Miki.

ABSTRACT

The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase is involved in the initial step of the 4HPA degradation pathway and catalyzes 4HPA hydroxylation to 3,4-dihydroxyphenylacetate. This enzyme consists of two components, an oxygenase (HpaB) and a reductase (HpaC). To understand the structural basis of the catalytic mechanism of HpaB, crystal structures of HpaB from Thermus thermophilus HB8 were determined in three states: a ligand-free form, a binary complex with FAD, and a ternary complex with FAD and 4HPA. Structural analysis revealed that the binding and dissociation of flavin are accompanied by conformational changes of the loop between beta5 and beta6 and of the loop between beta8 and beta9, leading to preformation of part of the substrate-binding site (Ser-197 and Thr-198). The latter loop further changes its conformation upon binding of 4HPA and obstructs the active site from the bulk solvent. Arg-100 is located adjacent to the putative oxygen-binding site and may be involved in the formation and stabilization of the C4a-hydroperoxyflavin intermediate.

Selected figure(s)

Figure 1.
FIGURE 1. A, crystal structure of HpaB homotetramer (the unliganded form). Each monomer is shown in a different color. B, a stereo view of the structure of HpaB monomer (the unliganded form). The N-terminal domain (Ala-2—Leu-138), the middle domain (Ala-139—Gly-266), the C-terminal domain (Asn-267—Tyr-456), and the -helical tail (Asn-457—Ala-481) are shown in yellow, cyan, orange, and magenta, respectively. The secondary structure elements are labeled. All figures were generated using the PyMOL program.

Figure 7.
FIGURE 7. A proposed catalytic mechanism of HpaB. (i) HpaB binds reduced FAD (FADH[2]), which is supplied from HpaC. (ii) FADH[2] reacts with an oxygen molecule and forms the C4a-hydroperoxyflavin intermediate. (iii) Substrate binds to the enzyme-C4a-hydroperoxyflavin complex. A hydroxyl group of the C4a-hydroperoxyflavin intermediate is introduced into the ortho position on the aromatic ring by electrophilic attack. (iv and v) The dienone form of the product is rearomatized, and DHPA and C4a-hydroxyflavin are formed. (vi) DHPA is released from the enzyme. (vii) Water is eliminated from the C4a-hydroxyflavin. (viii) Oxidized FAD is released from the enzyme and recycled to HpaC to be reduced for the next catalytic cycle.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 33107-33117) copyright 2007.

Figures were selected by an automated process.