PDBsum entry: 1jrq

Oxidoreductase

PDB id: 1jrq

Contents

Protein chains

718 a.a. *

Metals

_CA ×4

_CU ×2

Waters ×1394

* Residue conservation analysis

PDB id:

1jrq

Name:

Oxidoreductase

Title:

X-ray structure analysis of the role of the conserved tyrosine-369 in active site of e. Coli amine oxidase

Structure:

Copper amine oxidase. Chain: a, b. Synonym: tyramine oxidase, 2-phenylenthylamine oxidase. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Strain: dh5a. Cellular_location: periplasm. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.15Å R-factor: 0.195 R-free: 0.235

Authors:

J.M.Murray,C.R.Kurtis,W.Tambarajah,C.G.Saysell,C.M.Wilmot, M.R.Parsons,S.E.V.Phillips,P.F.Knowles,M.J.Mcpherson

Key ref:

J.M.Murray et al. (2001). Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential. Biochemistry, 40, 12808-12818. PubMed id: 11669617 DOI: 10.1021/bi011187p

Date:

14-Aug-01 Release date: 21-Nov-01

Protein chains

P46883  (AMO_ECOLI) -  Primary amine oxidase

Seq: 757 a.a.

Struc: 718 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.4.3.21 - Primary-amine oxidase.
• Reaction: RCH₂NH₂ + H₂O + O₂ = RCHO + NH₃ + H₂O₂

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

 Gene Ontology (GO) functional annotation 

• Cellular component: periplasmic space (1 term)
• Biological process: oxidation reduction (2 terms)
• Biochemical function: oxidoreductase activity (5 terms)

reference

DOI no: 10.1021/bi011187p

Biochemistry 40:12808-12818 (2001)

PubMed id: 11669617

Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential.

J.M.Murray, C.R.Kurtis, W.Tambyrajah, C.G.Saysell, C.M.Wilmot, M.R.Parsons, S.E.Phillips, P.F.Knowles, M.J.McPherson.

ABSTRACT

Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme. The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms. One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ. To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine. We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F. Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site.