Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100

Recent advances in structure and function of cytosolic IMP-GMP specific 5′nucleotidase II (cN-II)

Cytosolic 5′nucleotidase II (cN-II) catalyses both the hydrolysis of a number of nucleoside monophosphates (e.g., IMP + H2O→inosine + Pi), and the phosphate transfer from a nucleoside monophosphate donor to the 5′position of a nucleoside acceptor (e.g., IMP + guanosine →inosine + GMP). The enzyme protein functions through the formation of a covalent phosphoenzyme intermediate, followed by the phosphate transfer either to water (phosphatase activity) or to a nucleoside (phosphotransferase activity). It has been proposed that cN-II regulates the intracellular concentration of IMP and GMP and the production of uric acid. The enzyme might also have a potential therapeutic importance, since it can phosphorylate some anti-tumoral and antiviral nucleoside analogues that are not substrates of known kinases. In this review we summarise our recent studies on the structure, regulation and function of cN-II. Via a site-directed mutagenesis approach, we have identified the amino acids involved in the catalytic mechanism and proposed a structural model of the active site. A series of in vitro studies suggests that cN-II might contribute to the regulation of 5-phosphoribosyl-1-pyrophosphate (PRPP) level, through the so-called oxypurine cycle, and in the production of intracellular adenosine, formed by ATP degradation

Molecular Dynamics Simulation Study and Hybrid Pharmacophore Model Development in Human LTA4H Inhibitor Design

Human leukotriene A4 hydrolase (hLTA4H) is a bi-functional enzyme catalyzes the hydrolase and aminopeptidase functions upon the fatty acid and peptide substrates, respectively, utilizing the same but overlapping binding site. Particularly the hydrolase function of this enzyme catalyzes the rate-limiting step of the leukotriene (LT) cascade that converts the LTA4 to LTB4. This product is a potent pro-inflammatory activator of inflammatory responses and thus blocking this conversion provides a valuable means to design anti-inflammatory agents. Four structurally very similar chemical compounds with highly different inhibitory profile towards the hydrolase function of hLTA4H were selected from the literature. Molecular dynamics (MD) simulations of the complexes of hLTA4H with these inhibitors were performed and the results have provided valuable information explaining the reasons for the differences in their biological activities. Binding mode analysis revealed that the additional thiophene moiety of most active inhibitor helps the pyrrolidine moiety to interact the most important R563 and K565 residues. The hLTA4H complexes with the most active compound and substrate were utilized in the development of hybrid pharmacophore models. These developed pharmacophore models were used in screening chemical databases in order to identify lead candidates to design potent hLTA4H inhibitors. Final evaluation based on molecular docking and electronic parameters has identified three compounds of diverse chemical scaffolds as potential leads to be used in novel and potent hLTA4H inhibitor design

Structural Insights into the Inhibition of Cytosolic 5′-Nucleotidase II (cN-II) by Ribonucleoside 5′-Monophosphate Analogues

Cytosolic 5′-nucleotidase II (cN-II) regulates the intracellular nucleotide pools within the cell by catalyzing the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates. Beside this physiological function, high level of cN-II expression is correlated with abnormal patient outcome when treated with cytotoxic nucleoside analogues. To identify its specific role in the resistance phenomenon observed during cancer therapy, we screened a particular class of chemical compounds, namely ribonucleoside phosphonates to predict them as potential cN-II inhibitors. These compounds incorporate a chemically and enzymatically stable phosphorus-carbon linkage instead of a regular phosphoester bond. Amongst them, six compounds were predicted as better ligands than the natural substrate of cN-II, inosine 5′-monophosphate (IMP). The study of purine and pyrimidine containing analogues and the introduction of chemical modifications within the phosphonate chain has allowed us to define general rules governing the theoretical affinity of such ligands. The binding strength of these compounds was scrutinized in silico and explained by an impressive number of van der Waals contacts, highlighting the decisive role of three cN-II residues that are Phe 157, His 209 and Tyr 210. Docking predictions were confirmed by experimental measurements of the nucleotidase activity in the presence of the three best available phosphonate analogues. These compounds were shown to induce a total inhibition of the cN-II activity at 2 mM. Altogether, this study emphasizes the importance of the non-hydrolysable phosphonate bond in the design of new competitive cN-II inhibitors and the crucial hydrophobic stacking promoted by three protein residues

Structural basis for substrate specificity of the human mitochondrial deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3 amp; 8242; monophosphate, thymidine 5 amp; 8242; monophosphate, deoxyuridine 5 amp; 8242; monophosphate, uridine 5 amp; 8242; monophosphate, deoxyguanosine 5 amp; 8242; monophosphate, uridine 2 amp; 8242; monophosphate, and the 5 amp; 8242; monophosphate of the nucleoside analog 3 amp; 8242; deoxy 2 amp; 8242;3 amp; 8242; didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme s specificity for the deoxyribo form of nucleoside 5 amp; 8242; monophosphates is due to Ile 133, Phe 49, and Phe 102, which surround the 2 amp; 8242; position of the sugar and cause an energetically unfavorable environment for the 2 amp; 8242; hydroxyl group of ribonucleoside 5 amp; 8242; monophosphates. The close binding of the 3 amp; 8242; hydroxyl group of nucleoside 5 amp; 8242; monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzym

Structural Basis for Substrate Specificity of the Human Mitochondrial Deoxyribonucleotidase

The human mitochondrial deoxyribonucleotidase catalyzes the dephosphorylation of thymidine and deoxyuridine monophosphates and participates in the regulation of the dTTP pool in mitochondria. We present seven structures of the inactive D41N variant of this enzyme in complex with thymidine 3'-monophosphate, thymidine 5'-monophosphate, deoxyuridine 5'-monophosphate, uridine 5'-monophosphate, deoxyguanosine 5'-monophosphate, uridine 2'-monophosphate, and the 5'-monophosphate of the nucleoside analog 3'-deoxy 2'3'-didehydrothymidine, and we draw conclusions about the substrate specificity based on comparisons with enzyme activities. We show that the enzyme's specificity for the deoxyribo form of nucleoside 5'-monophosphates is due to Ile-133, Phe-49, and Phe-102, which surround the 2' position of the sugar and cause an energetically unfavorable environment for the 2'-hydroxyl group of ribonucleoside 5'-monophosphates. The close binding of the 3'-hydroxyl group of nucleoside 5'-monophosphates to the enzyme indicates that nucleoside analog drugs that are substituted with a bulky group at this position will not be good substrates for this enzyme

Crystal structures of the mitochondrial deoxyribonucleotidase in complex with two specific inhibitors

Monophosphate nucleotidases are enzymes that dephosphorylate nucleotides to their corresponding nucleosides. They play potentially important roles in controlling the activation of nucleotide-based drugs targeted against viral infections or cancer cells. The human mitochondrial deoxyribonucleotidase (dNT-2) dephosphorylates thymidine and deoxyuridine monophosphates. We describe the high resolution structures of the dNT-2 enzyme in complex with two potent nucleoside phosphonate inhibitors, (S)-1-[2'-deoxy-3',5'-O-(1-phosphono) benzylidene-beta-d-threo-pentofuranosyl]thymine (DPB-T) at 1.6-A resolution and (+/-)-1-trans-(2-phosphonomethoxycyclopentyl)uracil (PMcP-U) at 1.4-A resolution. The mixed competitive inhibitor DPB-T and the competitive inhibitor PMcP-U both bind in the active site of dNT-2 but in distinctly different binding modes, explaining their different kinetics of inhibition. The pyrimidine part of the inhibitors binds with very similar hydrogen bond interactions to the protein but with their phosphonate moieties in different binding sites compared with each other and to the previously determined position of phosphate bound to dNT-2. Together, these phosphate/phosphonate binding sites describe what might constitute a functionally relevant phosphate entrance tunnel to the active site. The structures of the inhibitors in complex with dNT-2, being the first such complexes of any nucleotidase, might provide important information for the design of more specific inhibitors to control the activation of nucleotide-based drugs

Crystal structures of human and murine deoxyribonucleotidases: Insights into recognition of substrates and nucleotide analogues

Cytosolic 5'(3')-deoxyribonucleotidase (cdN) and mitochondrial 5'(3')-deoxyribonucleotidase (mdN) catalyze the dephosphorylation of deoxyribonucleoside monophosphates and regulate dTTP formation in cytosol and mitochondria, protecting DNA replication from imbalanced precursor pools. They can also interfere with the phosphorylation-dependent activation of nucleoside analogues used in anticancer and antiviral treatment. To understand the relatively narrow substrate specificity of these two enzymes and their ability to use nucleotide analogues as substrates, we determined the crystal structures of human cdN in complex with deoxyuridine, murine cdN in complex with dUMP and dGMP, and human mdN in complex with the nucleotide analogues AZTMP and BVdUMP. Our results show that the active site residues Leu45 and Tyr65 in cdN form a more favorable binding surface for purine nucleotides than the corresponding Trp75 and Trp76 in mdN, explaining why cdN has higher activity for purine nucleotides than does mdN. The molecular interactions of mdN with AZTMP and BVdUMP indicate why these nucleotide analogues are poorer substrates as compared with the physiological substrate, and they provide a structural rationale for the design of drugs that are less prone to inactivation by the deoxyribonucleotidases. We suggest that introduction of substituents in the 3'-position may result in nucleoside analogues with increased resistance to dephosphorylation