Functional relevance of the internal hydrophobic cavity of urate oxidase

Abstract

a b s t r a c tUrate oxidase from Aspergillus flavus is a 135 kDa homo-tetramer which has a hydrophobic cavity buried within each monomer and located close to its active site. Crystallographic studies under moderate gas pressure and high hydrostatic pressure have shown that both gas presence and high pressure would rigidify the cavity leading to an inhibition of the catalytic activity. Analysis of the cavity volume variations and functional modifications suggest that the flexibility of the cavity would be an essential parameter for the active site efficiency. This cavity would act as a connecting vessel to give flexibility to the neighboring active site, and its expansion under pure oxygen pressure reveals that it might serve as a transient reservoir on its pathway to the active site.

On the Permeation by Dioxygen of Urate Oxidase from Aspergillus flavus in Complex with Xanthine Anion: Dioxygen Pathways and a Portrait of the Enzyme Cavities from Molecular Dynamics Simulations in Water Solution

“…It was observed that a moderate pressure of Xe gas induces a small contraction of the active site and a small expansion of the adjacent hydrophobic cavity, with Xe atoms populating the latter. With pressure exerted by O 2 gas, a similar phenomenon was observed, but no O 2 could be detected inside the above hydrophobic cavity, while a single O 2 molecule was found at the active center, over the substrate, on the internal protein side . It was also observed that high hydrostatic pressure has the reverse effect, contracting the active site, while expanding the nearby hydrophobic cavity.…”

“…On these bases, a first model for MD was built with a molecule of O 2 (represented at VdW size in red color in Fig. ) above 5 , toward the inside of the homotetramer, as indicated by the X‐ray diffraction studies . This model was used to investigate how O 2 can leave the protein under an accelerated form of MD, where expulsion of O 2 is aided by an external randomly oriented force (random acceleration MD, in short RAMD , as detailed below).…”

“…Lack of any indication, in the above studies, as to how O 2 could attain the active site of UOX, and speculations about the dynamic behavior of UOX cavities from static observations only , stimulated us to undertake an investigation of the dynamics of the complex of UOX with a uric acid mimics in the presence of O 2 . This endeavor places itself in a wider scenario, that of aerobic life, which, from the first appearance of O 2 in the world, has evolved to encompass nearly all living species, leaving anaerobic life to specialized niches only.…”

Determinants of neuroglobin plasticity highlighted by joint coarse-grained simulations and high pressure crystallography

“…HPMX allows to determine the most flexible parts of a protein in relation with its internal cavity modifications. The analyses of protein structures determined under high hydrostatic pressure have revealed that the main pressure-induced structural effects occur on the volumes of cavities which are reduced 32–35 . Coarse-grained simulations allow to investigate the mechanical properties of the proteins, in particular those of the frontier residues lining internal cavities, and highlight their mechanical nucleus 36–40 .…”

Direct Evidence for a Peroxide Intermediate and a Reactive Enzyme–Substrate–Dioxygen Configuration in a Cofactor‐free Oxidase

“…The position of O 2 observed here is consistent with that of room-temperature O 2 -pressurization experiments. [11] Peroxide breakdown also brings about a reorganization of the solvent around the organic moiety with the appearance of a solvent molecule (W2) bound to the O8 atom of the substrate. Raman analysis shows that peroxide rupture causes the 605 cm −1 ‘signature band’ to selectively decrease in a dose-dependent manner (Figure 4 b,c) consistent with our QM/MM calculations that assign this band to the stretching of the C5=Op1 bond.…”