Aldo-keto reductases in which the conserved catalytic histidine is substituted

Abstract

Aldo-keto reductases (AKRs) are a major superfamily of monomeric NADPH-dependent carbonyl oxidoreductases. They are characterized by an (α/β)₈-barrel structure, which at its base contains a conserved catalytic tetrad of Tyr, Lys, His and Asp. Two AKR subfamilies contain other residues substituted for the catalytic His and perform different functions. First, the steroid 5β-reductase (AKR1D1), which reduces CC double bonds instead of carbonyl groups, has a Glu substituted for His. Second, the Kvβ subunits (AKR6A3, AKR6A5 and AKR6A9) which modulate opening of the voltage-gated potassium channel (Kv1) by oxidizing NADPH, have an Asn substituted for the His. Previously, we noted that conserved catalytic residues in AKRs perform similar functions in the short-chain dehydrogenases (SDRs). With the availability of crystal structures of AKR1D1 and two SDRs that catalyze double-bond reduction reactions, Digitalis steroid 5β-reductase and 2,4-dienoyl-CoA reductase, we have compared their active sites to outline the features that govern whether 1,2-, 1,4- or 1,6-hydride transfer occurs.

Introduction

Two of the most prominent NAD(P)(H)-dependent oxidoreductases are the short-chain dehydrogenase reductases (SDRs) and the aldo-keto reductases (AKRs) [1], [2], [3], [4], [5]. These two enzyme superfamilies adopt different protein folds but share common endogenous substrates, e.g., steroid hormones, prostaglandins, and lipid aldehydes, as well as exogenous substrates such as xenobiotics, including aldehydes, ketones, and carbonyl containing drugs. Despite the differing structures of SDRs and AKRs, these enzyme superfamilies share common features of catalytic mechanism. The SDR active site contains a YXX(S)K motif, while the AKR active site contains conserved residues D50, Y55, K84, and H117 [4], [5], [6], [7], [8], [9], [10], [11]. Superposition of the active site residues of the SDR 3α(20β)-hydroxysteroid dehydrogenase (HSD) from Streptomyces hydrogenas[7] and rat 3α-hydroxysteroid dehydrogenase (AKR1C9) [11] shows that the general positions of the conserved tyrosine and lysine residues are generally similar relative to the si and re face of the nicotinamide ring of the cofactor, which donates either the 4-pro-S or 4-pro-R hydride in the SDR or AKR reactions, respectively (Fig. 1). It was also noted that the Ser in 3α(20β)-HSD and the Nɛ2 atom of His in AKR1C9 are both within hydrogen bonding distance of a water molecule that is displaced by the substrate carbonyl, suggesting that these residues play a facilitatory role in catalysis [7], [12]. It is apparent that the two enzyme superfamilies have convergently evolved to a common catalytic mechanism in which the tyrosine residue serves as a general acid–base [11]. In the SDRs the pKa of the Tyr is likely lowered by interaction with the nearby lysine residue, and the conserved Ser donates a hydrogen bond to the substrate carbonyl group (Fig. 2). In the AKRs, the Tyr acts a general base due to deprotonation by the lysine, but acts as a general acid by participating in a proton relay with the conserved His (Fig. 2) [13].

In the AKR superfamily, there are two subfamilies in which the conserved catalytic histidine residue is substituted [5]. In the AKR1D subfamily, which contains the steroid 5β-reductases, H117 is substituted by glutamic acid. In the AKR6A family, which contains the β-subunit Kvβ of the potassium channel Kv1, H117 is substituted by asparagine. Recently determined crystal structures of these AKRs have led to mechanistic proposals concerning the respective functions of these substituted residues [14]. These proposals are supported by comparisons to SDRs.