3cnj

From Proteopedia

Cholesterol oxidase from Streptomyces sp. F359W mutant (0.95A)

PDB ID 3cnj

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Structural highlights

3cnj is a 1 chain structure with sequence from Streptomyces sp. SA-COO. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 0.95Å
Ligands:	,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

CHOD_STRS0 Bifunctional enzyme that catalyzes the oxidation of the 3-beta-hydroxy group of cholesterol and the isomerization of the double bond of the resulting product.

Evolutionary Conservation

Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.

Publication Abstract from PubMed

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The K mO 2 increases from 300 +/- 35 microM for the wild-type enzyme to 617 +/- 15 microM for the F359W mutant. The k cat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase.,Chen L, Lyubimov AY, Brammer L, Vrielink A, Sampson NS Biochemistry. 2008 Apr 15;. PMID:18410129^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Chen L, Lyubimov AY, Brammer L, Vrielink A, Sampson NS. The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase. Biochemistry. 2008 Apr 15;. PMID:18410129 doi:10.1021/bi800228w