Distortion of flavin geometry is linked to ligand binding in cholesterol oxidase

Abstract

Two high-resolution structures of a double mutant of bacterial cholesterol oxidase in the presence or absence of a ligand, glycerol, are presented, showing the trajectory of glycerol as it binds in a Michaelis complex-like position in the active site. A group of three aromatic residues forces the oxidized isoalloxazine moiety to bend along the N5-N10 axis as a response to the binding of glycerol in the active site. Movement of these aromatic residues is only observed in the glycerol-bound structure, indicating that some tuning of the FAD redox potential is caused by the formation of the Michaelis complex during regular catalysis. This structural study suggests a possible mechanism of substrate-assisted flavin activation, improves our understanding of the interplay between the enzyme, its flavin cofactor and its substrate, and is of use to the future design of effective cholesterol oxidase inhibitors.

Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

“…Consistent with this, the pK a of Glu 109 , which is in an otherwise highly hydrophobic environment, is calculated to be ϳ9 by the PROPKA server (38). The ␥-carboxylate also hydrogen bonds via a water molecule to Glu 13 and His 40 , both putative catalytic residues, as discussed later. Five of eight hydrogen bonds made by the ␣-and ␤-carboxylates of citrate are to lysine or arginine side chains: Lys 198 , Lys 259 , and Arg…”

“…Additional interactions with the other face (the Si-face) at its two extremities then cause bending about an axis between the C5 and N3 atoms. At one end of the isoalloxazine system, the pyrimidine ring occupies a tight pocket formed by His 40 , Glu 109 , the protein backbone, and two ordered waters. At the other end the primary contact of the hydroxybenzyl ring is with the citrate ion, which occupies the substrate binding pocket, as described below.…”

“…This strongly supports the validity of the modeling and leads to a proposed reaction mechanism, described below. In particular, the C1 hydrogen is only 2.1 Å from C5 of F 420 , the recipient of the hydride ion transferred in the redox reaction, and the O1 hydroxyl is in hydrogen-bond distance to NE1 of Trp 44 , NE2 of His 40 , and OE2 of Glu 13 . Trp 44 can only act as a hydrogen bond donor, whereas His 40 is expected to act as a proton acceptor through NE2, as its ND1 atom is hydrogen-bonded to the main chain CAO of Gln 42 and is, thus, protonated.…”

“…This may also account for the more pronounced pyridine/hydroxybenzyl bend seen in FGD1 compared with Adf. Substrate binding has been shown to assist catalysis by promoting bending of the oxidized isoalloxazine moiety in another flavin-dependent enzyme, cholesterol oxidase (40).…”

Functional modulation of a protein folding landscape via side-chain distortion

“…In the case of αLP and its homologs, strain energy is used to extend functional lifetime, but we envision side-chain distortion also being relevant for other important macromolecular functions, such as allostery and catalysis. Although the functional importance of distortion in enzyme substrates (28)(29)(30) and cofactors (31)(32)(33)(34) has long been appreciated, and recent analysis points to conserved peptide bond distortions (35), the observation of side-chain distortion and the discovery that it can play a significant role in modulating energetic landscapes to provide biologically important advantages is quite unique. This study identifies an unanticipated challenge: the need to observe structurally subtle yet functionally significant covalent distortions to fully understand the energetic forces acting on proteins and their impact on function.…”