Structure of a xenon derivative ofEscherichia colicopper amine oxidase: confirmation of the proposed oxygen-entry pathway

Abstract

The mechanism of molecular oxygen entry into the buried active site of the copper amine oxidase family has been investigated in several family members using biochemical, structural and in silico methods. These studies have revealed a structurally conserved-sandwich which acts as a hydrophobic reservoir from which molecular oxygen can take several species-specific preferred pathways to the active site. Escherichia coli copper amine oxidase (ECAO) possesses an extra N-terminal domain that lies close to one entrance to the-sandwich. In order to investigate whether the presence of this domain alters molecular oxygen entry in this enzyme, xenon was used as a molecular oxygen binding-site probe. The resulting 2.5 Å resolution X-ray crystal structure reveals xenon bound in similar positions to those observed in xenon-derivative crystal structures of other family members, suggesting that the N-terminal domain does not affect oxygen entry and that the E. coli enzyme takes up oxygen in a similar manner to the rest of the copper amine oxidase family.

High-resolution crystal structure of copper amine oxidase fromArthrobacter globiformis: assignment of bound diatomic molecules as O2

“…However, hydrophilic oxygen-binding sites have also recently been discovered in cofactor-less oxidases and oxygenases using crystals under a high pressure of O 2 gas (Colloc'h et al, 2008) or crystals generated in a solution containing a halide ion, which is employed as a mimic of molecular oxygen (Gabison et al, 2010;Roeser et al, 2007;Steiner et al, 2010). In the present high-resolution structure of AGAO PG , the site that was presumed to be a consensus dioxygen (pre-)binding site (termed the 'anteroom'; Johnson et al, 2007) in previous structural studies using highly pressurized xenon gas (Lunelli et al, 2005;Duff et al, 2004;Johnson et al, 2007;Pirrat et al, 2008) is not occupied by an O 2 -like diatomic molecule. Similarly, none of the four assigned O 2 (or N 2 for Oxy3) molecules is found in the sites where Xe atoms are bound in AGAO (Supplementary Fig.…”

“…The Oxy2 binding site is present on a route from the central cavity (inland lake) to the active site, which has been proposed as one of the possible entry pathways for the substrate dioxygen (Wilce et al, 1997;Lunelli et al, 2005;McGrath et al, 2011;Duff et al, 2004). In the conservedsandwich, another candidate for the dioxygen pathway (Johnson et al, 2007;Pirrat et al, 2008), no bound dioxygen molecule was identified. On the other hand, Oxy3 (either O 2 or N 2 ) is located near the dimer interface and is surrounded by Arg490, Glu486*, Ala487*, Asp488*, Asp540* (where an asterisk denotes a residue from another subunit of the dimer) and diethyleneglycol [HO-(CH 2 CH 2 -O) n -H, n = 2] bound in this region (Fig.…”

“…S1b and S1c; Wilce et al, 1997;Airenne et al, 2005;Lunelli et al, 2005;Duff et al, 2004). Another possible pathway is from either end of the -sandwich in the D4 domain to the active site through the long hydrophobic region (Johnson et al, 2007;Pirrat et al, 2008). Common to both pathways, the end at the active site is around the anteroom, which has been proposed to be an oxygen pre-binding site (Johnson et al, 2007) and is composed mainly of hydrophobic residues (Ala400, Ala402, Phe435, Leu590, Asp605 and Val607 in AGAO; Supplementary Fig.…”

“…The prosthetic metal, Cu 2+ , is coordinated by the imidazole rings of three conserved histidine residues (His431, His433 and Hi592 in AGAO) by Cu 2+ -N bonds of about 2.0 Å in length. The entry route for molecular oxygen has been suggested to be either from the internal lake via a short polar channel or from either end of the -sandwich of the D4 domain through an internal hydrophobic region (Wilce et al, 1997;Lunelli et al, 2005;McGrath et al, 2011;Duff et al, 2004;Johnson et al, 2007;Pirrat et al, 2008).…”

Kinetic and Structural Analysis of Substrate Specificity in Two Copper Amine Oxidases from Hansenula polymorpha

“…All the residues that define the O 2 anteroom close to the copper are different between HPAO-1 and −2, but the nature of the area is still hydrophobic (Supporting Information Table S2). This is true in other CAOs, where the residues are not conserved but the hydrophobic nature of the pocket is maintained (22, 32, 42, 43). Hence, the kinetic and structural data suggest a modular structure for both HPAOs, in which changes at the amine substrate site are not propagated to the pocket where oxygen undergoes reaction.…”