Aldo-Keto Reductases (AKRs) are a superfamily of NAD(P)H linked oxidoreductases that are generally monomeric 34-37 kDa proteins present in all phyla. The superfamily consists of 15 families, which contains 151 members (www.med.upenn.edu/akr). Thirteen human AKRs exist that use endogenous substrates (sugar and lipid aldehydes, prostaglandins, retinals and steroid hormones), and in many instances they regulate nuclear receptor signaling. Exogenous substrates include metabolites implicated in chemical carcinogenesis: NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), polycyclic aromatic hydrocarbon trans-dihydrodiols, and aflatoxin dialdehyde. Promoter analysis of the human genes identifies common elements involved in their regulation which include osmotic response elements, antioxidant response elements, xenobiotic response elements, AP-1 sites and steroid response elements. The human AKRs are highly polymorphic, and in some instances single nucleotide polymorphisms (SNPs) of high penetrance exist. This suggests that there will be inter-individual variation in endogenous and xenobiotic metabolism which in turn affect susceptibility to nuclear receptor signaling and chemical carcinogenesis.

The M 2 ion channel protein of influenza A virus is essential for mediating protein-protein dissociation during the virus uncoating process that occurs when the virus is in the acidic environment of the lumen of the secondary endosome. The difficulty of determining the ion selectivity of this minimalistic ion channel is due in part to the fact that the channel activity is so great that it causes local acidification in the expressing cells and a consequent alteration of reversal voltage, V rev . We have confirmed the high proton selectivity of the channel (1.5-2.0 ؋ 10 6 ) in both oocytes and mammalian cells by using four methods as follows: 1) comparison of V rev with proton equilibrium potential; 2) measurement of pH in and V rev while Na The M 2 protein of influenza A virus is thought to function as an ion channel that permits protons to enter virus particles during virion uncoating in endosomes. In addition, in influenza virus-infected cells, the M 2 protein causes the equilibration of pH between the acidic lumen of the trans-Golgi network and the cytoplasm (reviewed in Refs. 1 and 2). The activity of the M 2 ion channel is inhibited by the antiviral drug amantadine (3-5). The mature M 2 protein consists of a 23-residue N-terminal extracellular domain, a single internal hydrophobic domain of 19 residues that acts as a transmembrane domain and forms the pore of the channel, and a 54-residue cytoplasmic tail (6). Chemical cross-linking studies showed the M 2 protein to exist minimally as a homotetramer (7-9). Statistical analysis of the ion channel activity of mixed oligomers also indicated that a homotetramer is the minimal active oligomeric form of the protein (10).Despite the small size of the active M 2 oligomer, several lines of evidence indicate that ion channel activity is intrinsic to the M 2 protein. First, ion channel activity has been observed in three different expression systems, Xenopus oocytes (3, 11, 12), mammalian cells (5, 13), and yeast (14). Second, M 2 channel activity has also been recorded in artificial lipid bilayers from a reconstituted peptide corresponding to the transmembrane domain of the M 2 protein (15) and from purified M 2 protein (16). Thus, due to its structural simplicity, the M 2 ion channel is a potentially useful model for the study of ion channels in general.Although a great deal of evidence indicates H ϩ is the biologically relevant ion for the role of M 2 protein in the life cycle of the influenza virus (1, 3, 17-22), other ions have been shown to be capable of flowing through the channel (12). In addition, the ion selectivity measured for the M 2 channel has been found to differ depending on whether the activity was measured in Xenopus oocytes or mammalian cells. When M 2 protein was expressed in oocytes, V rev was found to differ from the proton equilibrium potential, E Hϩ as [H ϩ ] out was varied (12). On the other hand, when M 2 protein was expressed in MEL cells, V rev was found to agree with E Hϩ (5). In a recent study (23), we found I Hϩ of the M 2 ion channel to...

AKR1D1 (steroid 5␤-reductase) reduces all ⌬ 4-3-ketosteroids to form 5␤-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an ␣,␤-unsaturated ketone by 5␤-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the ␤-face of a ⌬ 4 -3-ketosteroid yields a cis-A/B-ring configuration with an ϳ90°b end in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human ⌬ 120. The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.

Human steroid 5β-reductase (Aldo-keto Reductase 1D1) catalyzes the stereospecific NADPHdependent reduction of the C4-C5 double bond of Δ 4 -ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein.In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ 4 -ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5β-steroid metabolite present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the side-chain at C17 was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1 by contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.

The ⌬ 4 -3-ketosteroid functionality is present in nearly all steroid hormones apart from estrogens. The first step in functionalization of the A-ring is mediated in humans by steroid 5␣-or 5␤-reductase. Finasteride is a mechanism-based inactivator of 5␣-reductase type 2 with subnanomolar affinity and is widely used as a therapeutic for the treatment of benign prostatic hyperplasia. It is also used for androgen deprivation in hormonedependent prostate carcinoma, and it has been examined as a chemopreventive agent in prostate cancer. The effect of finasteride on steroid 5␤-reductase (AKR1D1) has not been previously reported. We show that finasteride competitively inhibits AKR1D1 with low micromolar affinity but does not act as a mechanism-based inactivator. The structure of the AKR1D1⅐NADP ؉ ⅐finasteride complex determined at 1.7 Å resolution shows that it is not possible for NADPH to reduce the ⌬ 1-2 -ene of finasteride because the cofactor and steroid are not proximal to each other. The C3-ketone of finasteride accepts hydrogen bonds from the catalytic residues Tyr-58 and Glu-120 in the active site of AKR1D1, providing an explanation for the competitive inhibition observed. This is the first reported structure of finasteride bound to an enzyme involved in steroid hormone metabolism.