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This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada.

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Aldose Reductase (2IKH)Aldose Reductase (2IKH)

spinqualitylabelsall models Human aldose reductase with bound ligand and NADPH Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

Information on aldose reductase is also available at Wikipedia

IntroductionIntroduction

is an enzyme that can reduce the aldehyde group of aldoses, aliphatic, aromatic aldehydes and some keto groups from aromatic and aliphatic ketones to their corresponding alcohol products using NADPH as a cofactor. Aldose reductase is most well known in the first step of the polyol pathway of glucose metabolism (Figure 1).

Figure 1. Polyol pathway of glucose metabolism. Adapted from Petrash JM. All in the family: aldose reductase and closely related aldo-keto reductases. Cell Mol Life Sci. 2004 Apr;61(7-8):737-49. PMID:15094999 doi:10.1007/s00018-003-3402-3

Polyol Pathway and DiabetesPolyol Pathway and Diabetes

As figure 1 shows, the polyol pathway involves the synthesis of fructose from glucose. The first step of the pathway is the production of sorbitol from glucose, catalyzed by aldose reductase and using NADPH as a reducing cofactor. The second step in the pathway is the production of fructose from sorbitol, catalyzed by sorbitol dehydrogenase using NAD+. Under normal blood glucose levels most glucose is metabolized through glycolysis or the pentose phosphate pathway while only a small amount of glucose is metabolized through the polyol pathway. Under the hyperglycemic conditions of diabetes the flux of glucose through the polyol pathway is increased. This causes osmotic and oxidative stress, which can cause pathological interferences with cytokine signalling, regulation of apoptosis, and activation of kinase cascades. For example, under increased glucose flux through the polyol pathway protein kinase C activivty increases, which causes smooth muscle cell proliferation of blood vessels in agreement with atherosclerosis. This explains estimates that 75-80% of adults with diabetes die from complications of atherosclerosis. Aldose reductase is also linked to long-term diabetic complications such as retinopathy, nephropathy, neuropathy, cataracts, and angiopathy. Aldose reductase inhibitors are possible beneficial treatment options for diabetes.

StructureStructure

Aldose reductase is a 36kDa aldo-keto reductase made of a single 315 amino acid residue polypeptide chain. It has a (β/α)8- structural motif made of 8 parallel β-strands connected to 8 peripheral α-helices running anti-parallel to the β-strands. Including the β-strands and α-helices of the TIM barrel, aldose reductase has a total of 10 . The catalytic active site is located at the C-terminal loop of the enzyme deeply buried inside the barrel core. This site consists of residues that are most likely involved in the catalytic reaction (including residues Tyr48, Lys77, His110). The cofactor is situated at the top of the barrel with the nicotinamide ring projecting down the center of the barrel and the pyrophosphate straddling the lip of the barrel. Trp111 and the nicotinamide moiety of NADPH interact with the head group of most . Hydrophobic contacts can be formed by the side-chains of Trp20, Val47, Trp79, and Trp219.

Aldose Reductase Structure and InhibitorsAldose Reductase Structure and Inhibitors

Most inhibitors that bind tightly to aldose reductase have a polar group, which is usually a carboxylate, that is attached to a hydrophobic core. Inhibitors bind with their polar head group oriented close to the pyridine ring and usually form hydrogen bonds with Tyr48, His110, and Tyr111. Hydrophobic interactions between the inhibitor and the residues that line the active site help to stabilize the ternary enzyme-coenzyme-inhibitor complex.

MechanismMechanism

The exact mechanism of the operation of the enzyme is under discussion. NADPH binds to the polypeptide first, followed by the substrate. The binding of NADPH induces a conformational change that involves a hinge-like movement of the surface loop (residues 213-217) so it covers part of the NADPH like a safety belt. NADPH donates a hydride ion to the carbonyl carbon of the aldehyde. The hydride transferred from NADPH to glucose comes from C-4 of the nicotinamide ring at the base of the hydrophobic cavity. Most likely then the transfer of a proton from one of the neighbouring acidic residues to the intermediately formed substrate ion occurs. Tyr48, His110, and Cys298 are all within a proper distance of C-4 to be potential proton donors. Evolutionary, thermodynamic, and molecular modeling evidence predicted that Tyr48 was the proton donor, which was later confirmed by mutagenesis studies. Hydrogen-bonding interactions between the phenolic hydroxyl group of Tyr48 and the ammonium side chain of Lys77 are thought to help facilitate hydride transfer. Lys 77 is salt linked to the carboxylate of Asp43. After the reaction occurs and the alcohol product has been released another conformational change occurs to release the NADP+. Kinetic studies have shown that the reorientation of the loop to release the NADP+ may be the rate-limiting step. Thus, disturbing the interactions that stabilize the coenzyme binding can have dramatic effects on the maximum rate of the reaction.