Oxygen Access to the Active Site of Cholesterol Oxidase through a Narrow Channel Is Gated by an Arg-Glu Pair

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 Å. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded antiparallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a ␤-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the ϳ8-Å movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

Section: Odcpo/hem13p Structurementioning

confidence: 99%

Crystal Structure of the Oxygen-dependant Coproporphyrinogen Oxidase (Hem13p) of Saccharomyces cerevisiae

Phillips¹,

Whitby

Warby³

et al. 2004

“…Although there is agreement on the physical mechanisms of the initial step(s) of electron transfer from the flavin to O 2 (12)(13)(14), key questions regarding the control of O 2 reactivity with the reduced flavin cofactor are still open. There are examples for channels in flavoproteins that guide O 2 to the reaction site (15)(16)(17). In this context, several questions emerge.…”

mentioning

confidence: 99%

O2 Reactivity of Flavoproteins

Saam

Rosini

Molla

et al. 2010

Self Cite

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O 2 in the protein framework of the flavoprotein D-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O 2 leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O 2 high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O 2 affinity. In this variant, the reactivity of the reduced enzyme with O 2 is decreased >100-fold and the turnover number ≈1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H ؉ from the product imino acid ‫؍‬NH 2 ؉ group bound on the flavin Reside to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.

“…A combination of structural and dynamic features like properly positioned positive charges, solvation of the active site, and protein breathing might play a role (12). The existence of (hydrophobic) oxygen channels from the surface to the active site has been proposed for several flavoenzymes, including the VAO members cholesterol oxidase (CO) (13) and alditol oxidase (AldO) (14).…”

mentioning

confidence: 99%

Identification of a Gatekeeper Residue That Prevents Dehydrogenases from Acting as Oxidases

Leferink

Fraaije

Joosten

et al. 2009

The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in L-galactono-␥-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or a Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala-113 3 Gly) resulted in a striking 400-fold increase in oxygen reactivity, whereas the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (k ox ‫؍‬ 3.5 ؋ 10 5 M ؊1 s ؊1 ), similar to most other flavoprotein oxidases. The Ala-113 3 Gly replacement does not change the reduction potential of the flavin but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme, Ala-113 acts as a gatekeeper, preventing oxygen from accessing the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family and is absent in members that act as oxidases.The flavoenzyme L-galactono-␥-lactone dehydrogenase (GALDH; 2 EC 1.3.2.3) catalyzes the terminal step in the biosynthesis of vitamin C (L-ascorbate) in plants. Besides producing this essential nutrient, GALDH is required for the accumulation of plant respiratory complex I (1). GALDH is an aldonolactone oxidoreductase that belongs to the vanillyl-alcohol oxidase (VAO) flavoprotein family (2). Members of this family share a two-domain folding topology with a conserved FAD binding domain and a cap domain that defines the substrate specificity (3). VAO family members include enzymes involved in carbohydrate metabolism and lignin degradation and enzymes that participate in the synthesis of antibiotics and alkaloids (4). Most VAO members contain a covalently tethered FAD and act as oxidases that use molecular oxygen to reoxidize the flavin, resulting in the production of hydrogen peroxide. In contrast to related aldonolactone oxidoreductases like L-gulono-␥-lactone oxidase from animals (5) and D-arabinono-␥-lactone oxidase from yeast (6), GALDH reacts poorly with molecular oxygen and contains non-covalently bound FAD (7). No crystal structure is available for the aldonolactone oxidoreductase subfamily, and little is known about the nature of the active site and the catalytic mechanism.GALDH is localized in the mitochondrial intermembrane space, where it feeds electrons into the respiratory chain. Its subcellular localization could provide a rationale why GALDH is a dehydrogenase and not, like related enzymes, an oxidase. The latter activity would result in high levels of mitochondrial hydrogen peroxide that promote GALDH inactivation (8) and induce aging, senescence, and cell death (9, 10). Furthermore, i...