Structural and Kinetic Determinants of Aldehyde Reduction by Aldose Reductase

Abstract

Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. Due to its ability to catalyze the formation of sorbitol from glucose during hyperglycemic and hypertonic stress, the aldose-reducing property of AR has been accepted as its main physiological and pathological function. Nonetheless, AR is a poor catalyst for glucose reduction and displays active-site properties unexpected of a carbohydrate-binding protein. We, therefore, examined the catalytic properties of AR with a series of naturally occurring aldehydes, compatible in their hydrophobicity to the large apolar active site of the enzyme. Our results show that recombinant human AR is an efficient catalyst for the reduction of medium- to long-chain unbranched saturated and unsaturated aldehydes. The enzyme displayed selective preference for saturated aldehydes, such as hexanal, and unsaturated aldehydes, such as trans-2-octenal and nonenal as well as their 4-hydroxy derivatives. Short-chain aldehydes such as propanal and acrolein were reduced less efficiently. Branched derivatives of acrolein or its glutathione conjugate (GS-propanal) were, however, reduced with high efficiency. In the absence of NADPH, the alpha, beta unsaturated aldehydes caused covalent modification of the enzyme. On the basis of electrospray mass spectrometric analysis of the wild-type and site-directed mutants of AR (in which the solvent exposed cysteines were individually replaced with serine), the site of modification was identified to be the active-site residue, Cys 298. The unsaturated aldehydes, however, did not modify the enzyme bound to NADPH and did not inactivate the enzyme during catalysis. Modeling studies indicate that the large hydrophobic active site of AR can accommodate a large number of aldehydes without changes in the structure of the binding site or movement of side chains. High hydrophobicity due to long alkyl chains or apolar substituents appears to stabilize the interaction of the aldehyde substrates with the enzyme. Apparently, such hydrophobic interactions provide substrate selectivity and catalytic efficiency of the order achievable by hydrogen bonding. Since several of the aldehydes reduced by AR are either environmental and pharmacological pollutants or products of lipid peroxidation, the present studies provide the basis of future investigations on the role of AR in regulating aldehyde metabolism particularly under pathological states associated with oxidative stress and/or aldehyde toxicity.

Aldose Reductase Mediates the Lipopolysaccharide-induced Release of Inflammatory Mediators in RAW264.7 Murine Macrophages

“…AR reduces one of the most abundant and toxic lipid aldehydes, 4-hydroxy-trans-2-nonenol (HNE), to 1, 4-dihydroxynonene (DHN) and its glutathione conjugate, GS-HNE, to GS-DHN (20,21). We have demonstrated that AR plays a pivotal role in the proliferation of vascular smooth mus-cle cells, apoptosis of vascular endothelial cells and restenosis of rat carotid artery (17,18,22).…”

Aldose Reductase Mediates Mitogenic Signaling in Vascular Smooth Muscle Cells

“…Extensive investigations show that AR is a broad specificity enzyme that catalyzes the reduction of a wide range of aldehydes. Its kinetic properties are optimized such that the energetics of substrate binding do not contribute to the overall catalytic efficiency of the enzyme (1,6). As a result, AR does not display predominant specificity for a unique substrate-product pair; rather, it can reduce several types of aldehydes with nearly equal efficiency.…”

“…As a result, AR does not display predominant specificity for a unique substrate-product pair; rather, it can reduce several types of aldehydes with nearly equal efficiency. Such behavior confers ideal properties to AR for the reduction of multiple aldehydes, such as the aldo-keto sugars and the aldehydes generated by lipid peroxidation (3)(4)(5)(6)(7)(8). The broad substrate specificity of the enzyme precludes its ready implication in a unique metabolic pathway and suggests that it may be recruited for tissue-specific use under a variety of metabolic contexts.…”

“…The reduction of glucose by AR is particularly significant during hyperglycemia, and increased flux of glucose via AR has been etiologically linked to the development of secondary diabetic complications (1,2). However, recent studies showing that AR is an excellent catalyst for the reduction of lipid peroxidation-derived aldehydes and their glutathione conjugates (3)(4)(5)(6)(7)(8) suggest that in contrast to its injurious role during diabetes, under normal glucose concentration, AR may be involved in protection against oxidative and electrophilic stress. The antioxidant role of AR is consistent with the observations that in a variety of cell types AR is up-regulated by oxidants such as hydrogen peroxide (9), lipid peroxidation-derived aldehydes (10,11), advanced glcosylation end products (12), and nitric oxide (13).…”

Mitogenic Responses of Vascular Smooth Muscle Cells to Lipid Peroxidation-derived Aldehyde 4-Hydroxy-trans-2-nonenal (HNE)

“…In most cases, GS-DHN represents 50% of the total conjugate. We have also found that treatment with AR inhibitors prevents the reduction of the conjugate (37,41,64,65) and that in vitro recombinant AR displays high affinity for glutathione conjugates of unsaturated aldehydes (39,40,42). Indeed, for C3 to C9 aldehydes, glutathiolation increases the catalytic efficiency of the enzyme (39).…”