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A highly efficient and facile protocol for the selective reduction of carboxylic acid of Fenofibric acid to corresponding alcohol was developed. The selective reduction was carried out by activation of carboxylic acid by mixed anhydride followed by the reaction of sodium borohydride in presence of methanol. This is the first example of chemoselective reduction of carboxylic acid to alcohol in presence of a ketone without any external catalyst or ligand in a single step. The reaction offers wide applicability for the selective carboxylic group reduction methodology. The chemoselective reduction was demonstrated by the reduction of Fenofibric acid, an active metabolite of the drug Fenofibrate, to corresponding alcohol in excellent selectivity, yield, and purity.

Background: Glutamate (Glu) is of great interest in biomedical research. It is considered a biomarker in diabetes, which may potentially contribute to the development of autism in genetically vulnerable human populations, and it is found in relation to advanced glycation end products (AGEs) [1]. Additionally, Glu plays an active role in the function of ligand-gated ion channel glutamate receptors, chloride channels capable of filtering glutamate, as well as Potassium (K+)-channel [2]. Glu attains α [3] and β [4] crystal forms and Cβ-CH2 show asymmetric 1H signal pattern in NMR spectra. Objectives: The current study was undertaken to understand the signal patterns of Cβ-CH2 in Glu of the smallest dipeptide, Glycylglutamic Acid (GlyGlu), as well as the order, and planarity of the amide bond in the molecule. Materials and Methods: NMR spectra of GlyGlu were measured in D2O to deduce 1H and 13C chemical shifts and coupling constants. GlyGlu was crystallized from MeOH and the structure was determined by single crystal X-ray diffraction techniques. Results: The sidechain of Glu in the dipeptide dissimilates the β form. The amino group of Gly (Glycine) is protonated and exhibits hydrogen bonding with the main chain carboxylate group of a symmetry-related Glu that is deprotonated in the crystal packing of GlyGlu. The deprotonated main chain carboxylate of Glu is also in hydrogen-bonding distance from the side chain carboxylic acid group that is in the protonated form of a symmetry-related Glu of the dipeptide. The Cβ-CH2 geminal protons on the side chain of Glu have different chemical shifts and splitting pattern in 1H NMR reflecting their dissymmetric environment. Conclusion: The results reported will be useful for monitoring changes that Glu and/or molecules in connection to Glu may undergo in in vivo, in situ, and in vitro conditions. This provides a valuable metric which will enable the examination of the metabolites relevant to the detection and diagnosis of disease or developmental conditions, as well as scrutinizing the effectiveness of treatment options.

Synthetic routes for the preparation of (i) isopropyl 2-(4-(4-chlorobenzyl)phenoxy)-2-methyl propanoate (Reduced Fenofibrate, Fibrane) (2) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate) (1) in a single step is established in good yield and purity under mild conditions. The newly synthesized derivative of Fenofibrate has been characterized by NMR and IR spectroscopy techniques. Selective conversion of biphenyl ketone moiety, in the presence of ester carbonyl group in Fenofibrate to its corresponding alkane can be performed by Pd catalyst reduction with hydrogen transfer agent ammonium formate.

Practical synthetic route for the formation of enantiomeric mixture of Isopropyl 2-(4-((4-chlorophenyl)(hydroxyl)methyl)phenoxy)-2-methylpropanoate (Fibratol 2a/b) from isopropyl 2-(4-(4-chlorobenzoyl)phenoxy)-2-methylpropanoate (Fenofibrate 1) has been developed. Method has also been established for the chiral separation of enantiomers of Fibratol 2a/b that is synthesized using the route mentioned above. The optical activity determined for enantiomerically separated Fibratol (2a) and Fibratol (2b) are −5.2° and 8.0° which reflect their ability to rotate plane polarized light counterclockwise (levo) and clockwise (dextro), respectively.

The peroxisome proliferator, WY 14,643 exhibits a pure non-competitive inhibition pattern in the aldehyde reduction and in alcohol oxidation activities of human Aldose reductase (hAR). Fluorescence emission measurements of the equilibrium dissociation constants, Kd, of oxidized (hAR•NADP+) and reduced (hAR•NADPH) holoenzyme complexes display a 2-fold difference between them. Kd values for the dissociation of WY 14,643 from the oxidized (hAR•NADP+•WY 14,643) and reduced (hAR•NADPH•WY 14,643) ternary complexes are comparable to each other. The ternary complex structure of hAR•NADP+•WY 14,643 reveals the first structural evidence of a fibrate class drug binding to hAR. These observations demonstrate how fibrate molecules such as WY 14,643, besides being valued as agonists for PPAR, also inhibit hAR.

Isopropyl 2-[4-(4-chlorobenzoyl)-phenoxy]-2-methylpropanoic acid and isopropyl 2-(4-chlorophenoxy)-2-methylpropanoate, also known as fenofibrate and isopropyl clofibrate, are hypolipidemic agents of the fibrate family. In a previously reported triclinic structure of fenofibrate (polymorph I) the methyl groups of the isopropyl moiety (iPr) are located symmetrically about the carboxylate group. We report a new monoclinic form (polymorph II) of fenofibrate and a first structural description of isopropyl clofibrate, and in these the methyl groups are placed asymmetrically about the carboxylate group. In particular the dihedral (torsion) angle between the hydrogen atom on the secondary C and the C atom of the carboxyl group makes a 2.74° angle about the ester O-C bond in the symmetric fenofibrate structure of polymorph I, whereas the same dihedral angle is 45.94° in polymorph II and -30.9° in the crystal structure of isopropyl clofibrate. Gas phase DFT geometry minimizations of fenofibrate and isopropyl clofibrate result in lowest energy conformations for both molecules with a value of about ± 30° for this same angle between the O=C-O-C plane and the C-H bond of the iPr group. A survey of crystal structures containing an iPr ester group reveals that the asymmetric conformation is predominant. Although the hydrogen atom on the secondary C atom of the isopropyl group is located at a comparable distance from the carbonyl oxygen in the symmetric and asymmetric fenofibrate (2.52 and 2.28 Å) and the isopropyl clofibrate (2.36 Å) structures, this hydrogen atom participates in a puckered five membered ring arrangement in the latter two that is unlike the planar arrangement found in symmetric fenofibrate (polymorph I). Polar molecular surface area (PSA) values indicate fenofibrate and isopropyl clofibrate are less able to act as acceptors of hydrogen bonds than their corresponding acid derivatives. Surface area calculations show dynamic polar molecular surface area (PSAd) values of the iPr esters of the fibrates are lower than those of their acids, implying that the fibrates have better membrane permeability and a higher absorbability and hence are better prodrugs when these agents need to be orally administered.

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Anthracyclines are an important reagent in many chemotherapy regimes for treating a wide range of tumors. One of the primary mechanisms of anthracycline action involves DNA damage caused by inhibition of topoisomerase II. Enzymatic detoxification of anthracycline is a major critical factor that determines anthracycline resistance. Natural product, daunorubicin a toxic analogue of anthracycline is reduced to less toxic daunorubicinol by the AKR1B10, enzyme, which is overexpressed in most cases of smoking associate squamous cell carcinoma (SCC) and adenocarcinoma. In addition, AKR1B10 was discovered as an enzyme overexpressed in human liver, cervical and endometrial cancer cases in samples from uterine cancer patients. Also, the expression of AKR1B10 was associated with tumor recurrence after surgery and keratinization of squamous cell carcinoma in cervical cancer and estimated to have the potential as a tumor intervention target colorectal cancer cells (HCT-8) and diagnostic marker for non-small-cell lung cancer. This article presents the mechanism of daunorubicin action and a method to improve the effectiveness of daunorubicin by modulating the activity of AKR1B10.

AKR1B10 is an aldose reductase (AR) homologue overexpressed in liver cancer and various forms of that enzyme in carcinomas catalyze the reduction of anticancer drugs, potential cytostatic drug, and dl-glyceraldehyde but do not catalyze the reduction of glucose. Kinetic parameters for wild-type and C299S mutant AKR1B10 indicate that substitution of serine for cysteine at position 299 reduces the affinity of this protein for dl-glyceraldehyde and enhances its catalytic activity. Fibrates suppress peroxisome proliferation and the development of liver cancer in human. Here we report the potency of fibrate-mediated inhibition of the carbonyl reduction catalyzed by wild-type and C299S mutant AKR1B10 and compare it with known AR inhibitors. Wild-type AKR1B10-catalyzed carbonyl reduction follows pure non-competitive inhibition kinetics using zopolrestat, EBPC or sorbinil, whereas fenofibrate, Wy 14,643, ciprofibrate and fenofibric acid follow mixed non-competitive inhibition kinetics. In contrast, catalysis of reaction by the C299S AKR1B10 mutant is not inhibited by sorbinil and EBPC. Despite these differences, the C299S AKR1B10 mutant still manifests kinetics similar to the wild-type protein with other fibrates including zopolrestat, fenofibrate, Wy 14,346, gemfibrozil and ciprofibrate that show mixed non-competitive inhibition kinetics. The reaction of the mutant AKR1B10 is inhibited by fenofibric acid, but manifests pure non-competitive inhibition kinetics that are different from those demonstrated for the wild-type enzyme.

Since the description of the synthetic chemical clofibrate in 1962, various derivatives of fibrates with a diversity of chemical structures have been developed. Several of these are used clinically to treat dyslipidemia because they are generally effective in lowering elevated plasma triglycerides and cholesterol. Studies suggest that several biochemical mechanisms underlie fibrate-mediated modulation of lipoprotein and related metabolites. These mechanisms are: 1) induced lipoprotein lipolysis; 2) induced hepatic fatty acid uptake and reduced hepatic triglyceride formation; 3) amplified removal of low density lipoprotein (LDL) particles; 4) reduced neutral lipid (cholesteryl ester and triglyceride) exchange between very low density lipoprotein (VLDL) and high density lipoprotein (HDL) resulting from decreased plasma levels of triglyceride-rich lipoprotein (TRL); and 5) increased HDL production and stimulation of reverse cholesterol transport. Recent studies of structure-based inhibitor design strategy revealed that an independent enzyme, aldose reductase (AR), is a target of fibrate activity, an additional biochemical mechanism. AR has been implicated as a major player in the development of diabetes and diabetic complications because of its ability to catalyze the conversion of glucose to sorbitol. This article discusses various targets of fibrate action, biochemical pathways and commonalities in potential molecular interactions.

Aldose reductase (AR) is implicated to play a critical role in diabetes and cardiovascular complications because of the reaction it catalyzes. Our data reveal that peroxisome proliferator WY 14,643, follows a pure non-competitive inhibition pattern in the aldehyde reduction activity as well as in the alcohol oxidation activity of AR. This finding communicates for the first time a novel feature of WY 14,643 in regulating AR activity. In addition, this observation indicates that AR, AR-like proteins and aldo-keto reductase (AKR) members may be involved in the WY 14,643 mechanism of action when it is administered as PPAR agonist.

Summary Protection from DNA invasion is afforded by restriction-modification systems in many bacteria. The efficiency of protection depends crucially on the relative expression levels of restriction versus methytransferase genes. This regulation is provided by a controller protein, named C protein. Studies of the BclI system in E. coli suggest that C.BclI functions as a negative regulator for M.BclI expression, implying that it plays a role in defense against foreign DNA during virus infection. C.BclI binds (K d = 14.3 nM) to a 2-fold symmetric C box DNA sequence that overlaps with the putative −35 promoter region upstream of the bclIM and bclIC genes. The C.BclI fold comprises five α helices: two helices form a helix-turn-helix motif, and the remaining three helices form the extensive dimer interface. The C.BclI-DNA model proposed suggests that DNA bending might play an important role in gene regulation, and that Glu27 and Asp31 in C.BclI might function critically in the regulation.

The NADPH-dependent reduction of glucose reaction that is catalyzed by Aldose Reductase (AR) follows a sequential ordered kinetic mechanism in which the co-factor NADPH binds to the enzyme prior to the aldehyde substrate. The kinetic/structural experiments have found a conformational change involving a hinge-like movement of a surface loop (residues 213-224) which is anticipated to take place upon the binding of the diphosphate moiety of NADPH. The reorientation of this loop, expected to permit the release of NADP+, represents the rate-limiting step of the catalytic mechanism. This study reveals: 1) The Translation/Libration/Screw (TLS) analysis of absolute B-factors of apo AR crystal structures indicates that the 212-224 loop might move as a rigid group. 2) Residues that make the flexible loop slide in the AR binary and ternary complexes. 3) The normalized B-factors separate this segment into three different clusters with fewer residues.

In this study, the selectivity and specificity of aldose reductase (AR) for glutathionyl aldehydes was examined. Relative to free aldehydes, AR was a more efficient catalyst for the reduction of glutathiolated aldehydes. Reduction of glutathionyl propanal [gammaGlu-Cys(propanal)-Gly] was more efficient than that of Gly-Cys(propanal)-Gly and gamma-aminobutyric acid-Cys(propanal)-Gly suggesting a possible interaction between alpha-carboxyl of the conjugate and AR. Two active site residues, Trp20 or Ser302, were identified by molecular modeling as potential sites of this interaction. Mutations containing tryptophan-to-phenylalanine (W20F) and serine-to-alanine (S302A) substitutions did not significantly affect reduction of free aldehydes but decreased the catalytic efficiency of AR for glutathiolated aldehydes. Combined mutations indicate that both Trp20 and Ser302 are required for efficient catalysis of the conjugates. The decrease in efficiency due to W20F mutation with glutathionyl propanal was not observed with gamma-aminobutyric-Cys(propanal)-Gly or Gly-Cys-(propanal)-Gly, indicating that Trp20 is involved in binding the alpha-carboxyl of the conjugate. The effect of the S302A mutation was less severe when gammaGlu-Cys(propanal)-Glu rather than glutathionyl propanal was used as the substrate, consistent with an interaction between Ser302 and Gly-3 of the conjugate. These observations suggest that glutathiolation facilitates aldehyde reduction by AR and enhances the range of aldehydes available to the enzyme. Because the N-terminal carboxylate is unique to glutathione, binding of the conjugate with the alpha-carboxyl facing the bottom of the alpha/beta-barrel may assist in the exclusion of unrelated peptides and proteins.

Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.

Comparisons of the structures of different mouse MHC class I molecules define how polymorphic residues determine the unique structural motif and atomic anchoring of their bound peptides. Here, Ted Hansen and colleagues speculate that quantitative differences in how class I molecules interact with peptide, β 2 -microglobulin and molecular chaperones that facilitate peptide loading might determine their relative participation in different pathways of antigen presentation.

Nascent class I molecules have been hypothesized to undergo a conformational change when they bind peptide based on the observation that most available antibodies only detect peptide-loaded class I. Furthermore recent evidence suggests that this peptide-facilitated conformational change induces the release of class I from association with transporter associated with antigen processing (TAP)/tapasin and other endoplasmic reticulum proteins facilitating class I assembly. To learn more about the structure of peptide-empty class I, we have studied mAb 64-3-7 that is specific for peptide-empty forms of L(d). We show here that mAb 64-3-7 detects a linear stretch of amino acids including principally residues 48Q and 50P. Furthermore, we demonstrate that the 64-3-7 epitope can be transferred to other class I molecules with limited mutagenesis. Interestingly, in the folded class I molecule residues 48 and 50 are on a loop connecting a beta strand (under the bound peptide) with the alpha(1) helix (rising above the ligand binding site). Thus it is attractive to propose that this loop is a hinge region. Importantly, the three-dimensional structure of this loop is strikingly conserved among class I molecules. Thus our findings suggest that all class I molecules undergo a similar conformational change in the loop around residues 48 and 50 when they associate with peptide.

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and β2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the β2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I α2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (k(cat)/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo - Fc)phi(calc)] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

A member of the aldo-keto reductase (AKR) protein superfamily, AKR1B10, is overexpressed in human liver cancers as well as in many adenocarcinoma cases due to smoking. AKR1B10 is also detected in instances of cervical and endometrial cancer in uterine cancer patients. In addition, AKR1B10 has been identified as a biomarker for non-small-cell lung cancer by a combined bioinformatics and clinical analysis. Furthermore, in breast cancer cells, fatty acid biosynthesis is regulated by AKR1B10. AKR1B10 contains 316 residues, shares 70% sequence identity with aldose reductase (AKR1B1) and has the conserved Cys residue at position 299. Carbonyl groups in some anticancer drugs and DL-glyceraldehyde are converted by AKR1B10 to their corresponding alcohols. The anticancer drug daunorubicin, which is currently used in the clinical treatment of various forms of cancer, is converted by AKR1B10 to daunorubicinol with a K-m and k(cat) of 1.1+/-0.18 mM and 1.4+/-0.16 min(-1), respectively. This carbonyl reducing activity of AKR1B10 decreases the anticancer effectiveness of daunorubicin. Similarly, kinetic parameters K-m and k(cat) (NADPH, DL-glyceraldehyde) for the reduction Of DL-glyceraldehyde by wild-type AKR1B10 are 2.2+/-0.2 mM and 0.71+/-0.05 sec(-1), respectively. Mutation of residue 299 from Cys to Set in AKR1B10 reduces the protein affinity for DL-glyceraldehyde and enhances AKR1B10's catalytic activity but overall catalytic efficiency is reduced. For DL-glyceraldehyde reduction that is catalyzed by the Cys299Ser mutant AKR1B10, K-m is 15.8+/-1.0 mM and k(cat) (NADPH, DL-glyceraldehyde) is 2.8+/-0.2 sec(-1). This implies that the substrate specificity of AKR1B10 is drastically affected by mutation of residue 299 from Cys to Set. In the present paper, we use this mutation in AKR1B10 to characterize a library of compounds regarding their different inhibitory potency on the carbonyl reducing activity of wild-type and the Cys299Ser mutant AKR1B10. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

International audience; Aldo-keto reductase AKR11C1 from Bacillus halodurans, a new member of aldo-keto reductase (AKR) family 11, has been characterized structurally and biochemically. The structures of the apo and NADPH bound form of AKR11C1 have been solved to 1.25 A and 1.3 A resolution, respectively. AKR11C1 possesses a novel non-aromatic stacking interaction of an arginine residue with the cofactor, which may favor release of the oxidized cofactor. Our biochemical studies have revealed an NADPH-dependent activity of AKR11C1 with 4-hydroxy-2,3-trans-nonenal (HNE). HNE is a cytotoxic lipid peroxidation product, and detoxification in alkaliphilic bacteria, such as B.halodurans, plays a crucial role in survival. AKR11C1 could thus be part of the detoxification system, which ensures the well being of the microorganism. The very poor activity of AKR11C1 on standard, small substrates such as benzaldehyde or DL-glyeraldehyde is consistent with the observed, very open active site lacking a binding pocket for these substrates. In contrast, modeling of HNE with its aldehyde function suitably positioned in the active site suggests that its elongated hydrophobic tail occupies a groove defined by hydrophobic side-chains. Multiple sequence alignment of AKR11C1 with the highly homologous iolS and YqkF proteins shows a high level of conservation in this putative substrate-binding site. We suggest that AKR11C1 is the first structurally characterized member of a new class of AKRs with specificity for substrates with long aliphatic tails.

Unlike the related fenofibrate molecule [Henry, Zhang, GaoBruckner (2003). Acta Cryst. E59, o699-o700], fenofibric acid {systematic name: 2-[4-(4-chlorobenzoyl)phenoxy]-2-methylpropanoic acid}, C17H15ClO4, contains a carboxylic acid moiety instead of an ester moiety. This polar moiety plays an important role in the formation of a rare acid-to-ketone hydrogen-bond-type packing interaction. The lack of an isopropyl group in fenofibric acid aligns the carboxyl group on the same side as the ketone carbonyl group; this conformation may play an important role in discrimination between the acid and the fenofibrate molecule in molecular recognition.

Expression of brain fatty acid-binding protein (B-FABP) is spatially and temporally correlated with neuronal differentiation during brain development. Isothermal titration calorimetry demonstrates that recombinant human B-FABP clearly exhibits high affinity for the polyunsaturated n-3 fatty acids alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, and for monounsaturated n-9 oleic acid (K(d) from 28 to 53 nm) over polyunsaturated n-6 fatty acids, linoleic acid, and arachidonic acid (K(d) from 115 to 206 nm). B-FABP has low binding affinity for saturated long chain fatty acids. The three-dimensional structure of recombinant human B-FABP in complex with oleic acid shows that the oleic acid hydrocarbon tail assumes a "U-shaped" conformation, whereas in the complex with docosahexaenoic acid the hydrocarbon tail adopts a helical conformation. A comparison of the three-dimensional structures and binding properties of human B-FABP with other homologous FABPs, indicates that the binding specificity is in part the result of nonconserved amino acid Phe(104), which interacts with double bonds present in the lipid hydrocarbon tail. In this context, analysis of the primary and tertiary structures of human B-FABP provides a rationale for its high affinity and specificity for polyunsaturated fatty acids. The expression of B-FABP in glial cells and its high affinity for docosahexaenoic acid, which is known to be an important component of neuronal membranes, points toward a role for B-FABP in supplying brain abundant fatty acids to the developing neuron.

Solution at 2.5-Å resolution of the three-dimensional structure of H-2Ldwith a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (β2m) and peptide. Consistent with the biological data that show an unusually weak association of Ldwith β2m, a novel orientation of the α1/α2 domains of Ldrelative to β2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ldantigen-binding cleft determine the unique motif of peptides that it binds. Ldhas no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ldand its peptide antigen. The unique structural interactions of Ldwith β2m and peptide could make Ldmore suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.