Metformin reverses hexokinase and 6-phosphofructo-1-kinase inhibition in skeletal muscle, liver and adipose tissues from streptozotocin-induced diabetic mouse

The present work describes the effects of metformin on hexokinase (HK) and phosphofructokinase (PFK) activities and localization in different tissues from streptozotocin-induced diabetic mice. Diabetic mice present lower HK and PFK activities (50%) in skeletal muscle, liver and adipose tissue, as compared with control (P < 0.05). Treatment with 250 mg/kg metformin reverses this pattern of enzyme inhibition with concomitant reversal of hyperglycemia and hypolactacidemia. Furthermore, the treatment increases the cytoskeleton-associated PFK activity in skeletal muscle; this activity has been described as an important mechanism for the enzyme activation. This effect might be due to the increased phosphorylation of serine residues in the enzyme, a modification which has been described to increase the interaction of PFK with f-actin. The current work supports the hypothesis that metformin hypoglycemic effects involve the activation of glycolysis through its regulatory enzymes, which may be potential targets for the development of new hypoglycemic drugs.     

Glucuronoxylomannan from Cryptococcus neoformans down-regulates the enzyme 6-phosphofructo-1-kinase of macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.     

Resveratrol decreases breast cancer cell viability and glucose metabolism by inhibiting 6-phosphofructo-1-kinase

Cancer cells are highly dependent on glycolysis to supply the energy and intermediates required for cell growth and proliferation. The enzyme 6-phosphofructo-1-kinase (PFK) is critical for glycolysis, and its activity is directly correlated with cellular glucose consumption. Resveratrol is a potential anti-tumoral drug that decreases glucose metabolism and viability in cancer cells. However, the mechanism involved in resveratrol-mediated anti-tumor activity is not entirely clear. In this work, it is demonstrated that resveratrol decreases viability, glucose consumption and ATP content in the human breast cancer cell line MCF-7. These effects are directly correlated with PFK inhibition by resveratrol in these cells. Moreover, resveratrol directly inhibits purified PFK, promoting the dissociation of the enzyme from fully active tetramers into less active dimers. This effect is exacerbated by known negative regulators of the enzyme, such as ATP and citrate. On the other hand, positive modulators that stabilize the tetrameric form of the enzyme, such as fructose-2,6-bisphosphate and ADP, prevent the inhibition of PFK activity by resveratrol, an effect not observed with increased pH. In summary, our results provide evidence that resveratrol directly inhibits PFK activity, therefore disrupting glucose metabolism and reducing viability in cancer cells.     

Fructose-2,6-bisphosphate counteracts guanidinium chloride-, thermal-, and ATP-induced dissociation of skeletal muscle key glycolytic enzyme 6-phosphofructo-1-kinase: A structural mechanism for PFK allosteric regulation

Rabbit muscle 6-phosphofructo-1-kinase (PFK) is the key glycolytic enzyme being regulated by diverse molecules and signals. This enzyme may undergo a reversible dissociation from a fully active homotetramer to a quite inactive dimer. There are evidences that some positive and negative modulators of PFK, such as ADP and citrate, may interfere with the enzyme oligomeric structure shifting the tetramer-dimer equilibrium towards opposite orientations, where the negative modulators favor the dissociation of tetramers into dimers and vice versa. PFK is allosterically inhibited by ATP at its physiological range of concentration, an effect counteracted by fructose-2,6-bisphosphate (F2,6BP). However, the structural molecular mechanism by which ATP and F2,6BP regulate PFK is hitherto demonstrated. The present paper aimed at demonstrating that either the ATP-induced inhibition of PFK and the reversion of this inhibition by F2,6BP occur through the same molecular mechanism, i.e., the displacement of the oligomeric equilibrium of the enzyme. This conclusion is arrived assessing the effects of ATP and F2,6BP on PFK inactivation through two distinct ways to dissociate the enzyme: (a) upon incubation at 50 °C, or (b) incubating the enzyme with guanidinium hydrochloride (GdmCl). Our results reveal that temperature- and GdmCl-induced inactivation of PFK prove remarkably more effective in the presence 5 mM ATP than in the absence of additives. On the other hand, the presence of 100 nM F2,6BP attenuate the effects of both high-temperature exposition and GdmCl on PFK, even in the simultaneous presence of 5 mM ATP. These data support the hypothesis that ATP shifts the oligomeric equilibrium of PFK towards the smaller conformations, while F2,6BP acts in the opposite direction. This conclusion leads to important information about the molecular mechanism by which PFK is regulated by these modulators.     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

Regulation of mammalian muscle type 6-phosphofructo-1-kinase and its implication for the control of the metabolism

Phosphofructokinase (PFK) is a major regulatory glycolytic enzyme and is considered to be the pacemaker of glycolysis. This enzyme presents a puzzling regulatory mechanism that is modulated by a large variety of metabolites, drugs, and intracellular proteins. To date, the mammalian enzyme structure has not yet been resolved. However, it is known that PFK undergoes an intricate oligomerization process, shifting among monomers, dimers, tetramers, and more complex oligomeric structures. The equilibrium between PFK dimers and tetramers is directly correlated with the enzyme regulation, because the dimer exhibits very low catalytic activity, whereas the tetramer is fully active. Several PFK ligands modulate the enzyme, favoring the formation of its dimers or tetramers. The present review integrates recent findings regarding the regulatory aspects of muscle type PFK and discusses their relation to the control of metabolism.     

Hypoxic regulation of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family (PFKFB-1-4) expression in vivo

When oxygen becomes limiting, cells shift primarily to a glycolytic mode for generation of energy. A key regulator of glycolytic flux is fructose-2,6-bisphosphate (F-2,6-BP), a potent allosteric regulator of 6-phosphofructo-1-kinase (PFK-1). The levels of F-2,6-BP are maintained by a family of bifunctional enzymes, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB or PFK-2), which have both kinase and phosphatase activities. Each member of the enzyme family is characterized by their phosphatase:kinase activity ratio (K:B) and their tissue-specific expression. Previous work demonstrated that one of the PFK-2 isozyme genes, PFKFB-3, was induced by hypoxia through the hypoxia-inducible factor-1 (HIF-1) pathway. In this study we examined the basal and hypoxic expression of three members of this family in different organs of mice. Our findings indicate that all four isozymes (PFKFB-1-4) are responsive to hypoxia in vivo. However, their basal level of expression and hypoxia responsiveness varies in the different organs studied. Particularly, PFKFB-1 is highly expressed in liver, heart and skeletal muscle, with the highest response to hypoxia found in the testis. PFKFB-2 is mainly expressed in the lungs, brain and heart. However, the highest hypoxia responses are found only in liver and testis. PFKFB-3 has a variable low basal level of expression in all organs, except skeletal muscle, where it is highly expressed. Most importantly, its hypoxia responsiveness is the most ample of all three genes, being strongly induced in the lungs, liver, kidney, brain, heart and testis. Further studies showed that PFKFB-1 and PFKFB-2 were highly responsive to hypoxia mimics such as transition metals, iron chelators and inhibitors of HIF hydroxylases, suggesting that the hypoxia responsiveness of these genes is also regulated by HIF proteins. In summary, our data demonstrate that PFK-2 genes are responsive to hypoxia in vivo, indicating a physiological role in the adaptation of the organism to environmental or localized hypoxia/ischemia.     

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.     

Targeting the Warburg Effect in cancer; relationships for 2-arylpyridazinones as inhibitors of the key glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3)

High-throughput screening of a small-molecule library identified a 5-triazolo-2-arylpyridazinone as a novel inhibitor of the important glycolytic enzyme 6-phosphofructo-2-kinase/2,6-bisphosphatase 3 (PFKFB3). Such inhibitors are of interest due to PFKFB3’s control of the important glycolytic pathway used by cancer cells to generate ATP. A series of analogues was synthesized to study structure-activity relationships key to enzyme inhibition. Changes to the triazolo or pyridazinone rings were not favoured, but limited-size substitutions on the aryl ring provided modest increases in potency against the enzyme. Selected analogues and literature-described inhibitors were evaluated for their ability to suppress the glycolytic pathway, as detected by a decrease in lactate production, but none of these compounds demonstrated such suppression at non-cytotoxic concentrations.     

C-terminal modification of 6-phosphofructo-1-kinase from Saccharomyces cerevisiae and its influence on enzyme structure and activity

Studies on limited proteolysis of 6-phosphofructo-1-kinase (Pfk-1) from Saccharomyces cerevisiae led to the suggestion that the C-terminal part of the alpha-subunit must contribute to the stabilisation of the octameric enzyme structure. To analyse the role of the C-terminus in vivo, the respective terminus of one of both types of subunits of Pfk-1 was sequentially truncated or extended. These modifications resulted in a decrease of the protein level of the mutated subunit and of the specific enzyme activity in the cell-free extract as well as in changes of the kinetic properties. Size exclusion HPLC demonstrated that the modified subunit is still able to assemble with the native counterpart generating an enzymatically active hetero-octamer. On the basis of our results we assume that the C-termini are important for the three-dimensional structure of the subunits determining their susceptibility to proteolysis and the ability to assembly to an active, oligomeric Pfk-1.     

Evidence for the activation of 6-phosphofructo-1-kinase by cAMP-dependent protein kinase in Aspergillus niger

Abstract The change from pentose phosphate pathway to glycolysis plays a significant role in the physiology of Aspergillus niger during the induction of citric acid accumulation. Evidence is shown for the importance of 6-phophofructo-1-kinase in this process since it is activated by phosphorylation. By incubating a purified active form of enzyme together with commercially available alkaline phosphatase, 6-phosphofructo-1-kinase activity was lost after a certain time suggesting that the enzyme was dephosphorylated. Inactive 6-phosphofructo-1-kinase could be isolated from the cells in the early stage of growth in a high citric acid yielding medium. The enzyme was 'in vitro' activated by isolated protein kinase in the presence of cAMP, ATP and Mg²⁺ ions. Additional evidence for covalent phosphorylation of inactive 6-phosphofructo-1-kinase was obtained by incubating both enzymes together with labelled [ γ −³²P]ATP. The activating enzyme was partially purified from A. niger mycelium.     

SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane

GTP phosphorylation of rough microsomes in vitro is limited to four integral membrane proteins. Two of these, a phosphoprotein (pp90) and a phosphoglycoprotein (pgp35) were purified as a complex with two nonphosphorylated membrane glycoproteins, gp25H and gp25L. The authenticity of this complex was confirmed using two different purification procedures and by coimmunoprecipitation. By immunofluorescence a reticulated cytoplasmic network was revealed for the proteins which was similar to that for Louvard et al. (Louvard, D., Reggio, H., and Warren, G. (1982) J. Cell Biol. 92, 92-107) marker antisera which also recognized purified pp90 on immunoblots. Amino acid sequencing of peptides derived from pgp35 identified this protein as SSR alpha, an endoplasmic reticulum constituent as identified by cross-linking of translocating nascent chains (G?rlich, D, Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990) J. Cell Biol. 111, 2283-2294). The sequence of gp25H was determined from cDNA clones and was identical with SSR beta identified by G?rlich et al. (1990) as being tightly bound to SSR alpha. Sequencing of gp25L revealed no similarity of the deduced sequence with other proteins. However, pp90 revealed a high degree of sequence identity with the Ca(2+)-binding protein, calreticulin. 45Ca2+ overlay studies indicated that pp90 bound Ca2+ and the name calnexin is proposed. Surprisingly, pgp25 (SSR alpha) also bound Ca2+ although gp25H (SSR beta) and gp25L did not. Triton X-114 partitioning of the integral membrane proteins of rough microsomes suggested that pgp35 (SSR alpha) and calnexin were major Ca(2+)-binding proteins of the endoplasmic reticulum membrane. We propose that the function of the complex is to regulate Ca(2+)-dependent retention mechanisms for luminal proteins of the endoplasmic reticulum.     

p-(Chloromercuri)benzenesulfonate binding by membrane proteins and the inhibition of water transport in human erythrocytes

The binding of [203Hg]-p-(chloromercuri)benzenesulfonate to the membrane proteins of human erythrocytes and erythrocyte ghosts was examined under conditions where binding to the bulk of membrane sulfhydryl groups was blocked by N-ethylmaleimide. Binding was essentially complete within 90 min when approximately 40 nmol was bound per milligram of membrane protein. This binding was correlated with the inhibition of water transport measured by an NMR technique. Maximal inhibition was observed with the binding of approximately 10 nmol of p-(chloromercuri)benzenesulfonate/mg of membrane protein. Under these conditions, both band 3 and band 4.5 bound 1 mol of inhibitor/mol of protein. In contrast to previous experiments, these results indicate that band 4.5 proteins as well as band 3 have to be considered as playing a role in water transport.     

Evidence that arginine-129 and arginine-145 are located within the heparin binding site of human antithrombin III

Arginyl residues of human antithrombin III have been implicated to involve in the heparin binding site [Jorgensen, A. M., Borders, C. L., & Fish, W. W. (1985) Biochem, J. 231, 59-63]. We have performed chemical modification of antithrombin with (p-hydroxyphenyl)glyoxal (HPG) in order to determine the locations of these arginine residues. Antithrombin was modified with 12 mM HPG in the absence and presence of heparin (2-fold by weight to antithrombin). In the absence of heparin, about 3-4 mol of arginines/mol of antithrombin were modified within 60 min, and the modification led to the loss of 95% of the inhibitor's heparin cofactor activity as well as heparin-induced fluorescence enhancement and 50% of its progressive inhibitory activity. In the presence of heparin, the extent of modification was diminished by 30% and modified antithrombin retained approximately 70% of its heparin cofactor activity. Peptide mapping and subsequent sequence analysis revealed that selective HPG modification occurred at Arg129 and Arg145 and that their modifications were protected upon binding of heparin to antithrombin. We conclude that Arg129 and Arg145 are situated within the heparin binding site of human antithrombin III.     

Structure of human alpha1-acid glycoprotein and its high-affinity binding site

Secondary and tertiary structures of human blood alpha(1)-acid glycoprotein, a member of the lipocalin family, have been studied for the first time by infrared and Raman spectroscopies. Vibrational spectroscopy confirmed details of the secondary structure and the structure content predicted by homology modeling of the protein moiety, i.e., 15% alpha-helices, 41% beta-sheets, 12% beta-turns, 8% bands, and 24% unordered structure at pH 7.4. Our model shows that the protein folds as a highly symmetrical all-beta protein dominated by a single eight-stranded antiparallel beta-sheet. Thermal dynamics in the range 20-70 degrees C followed by Raman spectroscopy and analyzed by principle component analysis revealed full reversibility of the protein motion upon heating dominated by decreasing of beta-sheets. Raman difference spectroscopy confirmed the proximity of Trp(122) to progesterone binding.     

The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site

Ehrlich's pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of dye molecules including trypan blue and trypan red to eliminate trypanosome infections in mice. The molecular structures of the dyes provided a starting point for the synthesis of suramin, which was developed and used as a trypanocidal drug in 1916 and is still in clinical use. Despite the biological importance of these dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of suramin and three related dyes in complex with pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (suramin, Ponceau S, acid blue 80, and benzothiazole-2,5-disulfonic acid) bind in the position of ADP/ATP at the active sites of the pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that suramin competitively inhibits PYKs from humans (muscle, tumor, and liver isoenzymes, K(i) = 1.1-17 μM), T. cruzi (K(i) = 108 μM), and L. mexicana (K(i) = 116 μM), all of which have similar active sites. Synergistic effects were observed when examining suramin inhibition in the presence of an allosteric effector molecule, whereby IC(50) values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for suramin and into the mode of action of the dye-like molecules used in Ehrlich's original experiments.     

Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins.

E6-AP is a 100-kDa cellular protein that mediates the interaction of the human papillomavirus type 16 and 18 E6 proteins with p53. The association of p53 with E6 and E6-AP promotes the specific ubiquitination and subsequent proteolytic degradation of p53 in vitro. We recently isolated a cDNA encoding E6-AP and have now mapped functional domains of E6-AP involved in binding E6, association with p53, and ubiquitination of p53. The E6 binding domain consists of an 18-amino-acid region within the central portion of the molecule. Deletion of these 18 amino acids from E6-AP results in loss of both E6 and p53 binding activities. The region that directs p53 binding spans the E6 binding domain and consists of approximately 500 amino acids. E6-AP sequences in addition to those required for formation of a stable ternary complex with E6 and p53 are necessary to stimulate the ubiquitination of p53. These sequences lie within the C-terminal 84 amino acids of E6-AP. The entire region required for E6-dependent ubiquitination of p53 is also required for the ubiquitination of an artificial E6 fusion protein.     

Identification of an epitope within human integrin alpha 6 subunit for the binding of autoantibody and its role in basement membrane separation in oral pemphigoid

Oral pemphigoid (OP) is a rare chronic autoimmune disease characterized by blisters and erosive lesions in the oral mucosa. We identified an epitope for the binding of OP autoantibodies within the integrin alpha6 subunit, by cloning four overlapping fragments (A, B, C, and D). Immunoperoxidase studies demonstrated that all of the fragments were present in the oral mucosa. Sera of 20 patients with active OP were studied. All sera bound to integrin alpha6 in DU145 cell lysate by immunoprecipitation and immunoblot assay. The same sera bound only to fragment A and its subfragment A2 on an immunoblot assay. The specificity of the binding was further characterized by blocking and cross-absorption studies. A 14-aa synthetic peptide A2.1, within fragment A2, bound to all the test sera. The sera in this study bound to only one epitope. Controls were sera samples from 10 healthy volunteers and 40 patients with other variants of mucous membrane pemphigoid and mAb GoH3 and BQ16 to integrin alpha6. Control sera did not bind to the full-length integrin alpha6 subunit nor any of the cloned fragments. The OP patient sera and immunoaffinity-purified OP sera, rabbit antisera against fragments A and A2, and mAb GoH3 produced basement membrane separation of oral mucosa in organ culture. This study identifies a peptide within the extracellular domain of integrin alpha6 molecule, to which Abs in the sera from patients with OP bind, and which may play an important role in the pathogenesis of OP.     

Phenylalanine(90) and phenylalanine(93) are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10

Human UDP-glucuronosyltransferase 1A10 has been identified as the major isoform involved in the biotransformation of a wide range of phenolic substrates, including native estrogens and their oxidized metabolites. Our recent studies point to the F(90)-M(91)-V(92)-F(93) amino acid motif of UGT1A10, which was identified using photoaffinity labeling followed by LC-MS/MS analysis, as a key determinant of the binding of phenolic substrates. In this report, we have evaluated the role of F(90), V(92), and F(93) in the recognition of estrogens by UGT1A10 using site-directed mutagenesis. Kinetic studies using five mutants revealed that F(90) and F(93) are critical residues for the recognition of all estrogen substrates. The substitution of F(90) with alanine totally abolished the activity of this enzyme toward all the estrogens investigated. Overall, sequential removal for the aromatic ring (F to L) and of the hydrophobic chain (F to A and V to A) from amino acids 90, 92, and 93 effectively alters estrogen recognition. This demonstrates that individual features of the native and hydroxylated estrogens determine the specific binding properties of the compound within the binding site of the human UGT1A10 and the mutants. The resulting activities are completely abolished, unchanged, increased, or decreased depending on the structures of both the mutant and the substrate. The novel identification of UGT1A10 as the major isoform involved in the glucuronidation of all estrogens and the discovery of the importance of the FMVF motif in the binding of steroids will help to elucidate the molecular mechanism of glucuronidation, resulting in the design of more effective estrogen-based therapies.