Genetically engineered analogs of ascomycin for nerve regeneration.

The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.

An FKBP12 binding assay based upon biotinylated FKBP12.

A binding assay was developed for measuring the affinity of FKBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase. The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with [3H]FK506 for FKBP12 sites on the plate. The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules.

Manipulation of polyketide biosynthesis for new drug discovery.

Modular polyketide synthases (PKS) are large multifunctional proteins which direct the condensation of activated short chain carboxylic acids into products of defined length and functionality using a dedicated set of active sites, or module, for each step in the polymerization. The structure of the product is directly related to the number, content and sequence of modules in a PKS. Technology is described which allows the rational manipulation of the biosynthesis of these compounds and enables the generation of specific novel polyketide structures. Examples of polyketide drugs whose structures may be manipulated using this technology are given.

Development and screening of a polyketide virtual library for drug leads against a motilide pharmacophore.

A virtual library of macrocyclic polyketide molecules was generated and screened to identify novel, conformationally constrained potential motilin receptor agonists ("motilides"). A motilide pharmacophore model was generated from the potent 6,9-enol ether erythromycin and known derivatives from the literature. The pharmacophore for each molecular conformation was a point in a distance-volume space based on presentation of the putative binding moieties. Two methods, one fragment based method and the other reaction based, were explored for constructing the polyketide virtual library. First, a virtual library was assembled from monomeric fragments using the CHORTLES language. Second, the virtual library was assembled by the in silico application of all possible polyketide synthase enzyme reactions to generate the product library. Each library was converted to low-energy 3D conformations by distance geometry and standard minimization methods. The distance-volume metric was calculated for low-energy conformations of the members of the virtual polyketide library and screened against the enol ether pharmacophore. The goal was to identify novel macrocycles that satisfy the pharmacophore. We identified three conformationally constrained, novel polyketide series that have low-energy conformations satisfying the distance-volume constraints of the motilide pharmacophore.

Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase.

A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

Utilization of enzymatically phosphopantetheinylated acyl carrier proteins and acetyl-acyl carrier proteins by the actinorhodin polyketide synthase.

The functional reconstitution of two purified proteins of an aromatic polyketide synthase pathway, the acyl carrier protein (ACP) and holo-ACP synthase (ACPS), is described. Holo-ACPs were enzymatically synthesized from coenzyme A and apo-ACPs using Escherichia coli ACPS. Frenolicin and granaticin holo-ACPs formed in this manner were shown to be fully functional together with the other components of the minimal actinorhodin polyketide synthase (act PKS), resulting in synthesis of the same aromatic polyketides as those formed by the act PKS in vivo. ACPS also catalyzed the transfer of acetyl-, propionyl-, butyryl-, benzoyl-, phenylacetyl-, and malonylphosphopantetheines to apo-ACPs from their corresponding coenzyme As, as detected by electrophoresis and/or mass spectrometry. A steady state kinetic study showed that acetyl-coenzyme A is as efficient an ACPS substrate as coenzyme A, with kcat and Km values of 20 min-1 and 25 microM, respectively. In contrast to acetyl-coenzyme A, enzymatically synthesized acetyl-ACPs were shown to be efficient substrates for the act PKS, indicating that acetyl-ACP is a chemically competent intermediate of aromatic polyketide biosynthesis. Together, these methods provide a valuable tool for dissecting the mechanisms and molecular recognition features of polyketide biosynthesis.

The catalytic mechanism and structure of thymidylate synthase.

Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.

Heterodimeric thymidylate synthases with C-terminal deletion on one subunit.

We have combined site-directed mutagenesis with the technique of reversible unfolding and subunit dissociation to construct heterodimeric thymidylate synthases that lack the C-terminal valine from only one subunit of the dimer. Removal of this residue either from both subunits of the dimer by mutagenesis (V316Am mutation) or from only one subunit by treatment with carboxypeptidase has been reported to result in an inactive enzyme (Carreras, C. W., Climie, S. C., and Santi, D. V. (1992) Biochemistry 31, 6038-6044; Aull, J.L., Loeble, R.B., and Dunlap. R.B. (1974) J. Biol. Chem. 249, 1167-1172). Arg-178 is an essential active site residue of thymidylate synthase that is donated from the opposing subunit of the dimer. The R178F-V316Am heterodimer was formed by the unfolding and refolding of a mixture of inactive R178F and V316Am mutants. This enzyme has one intact active site and was found to have half of the activity and the same Km values as wild-type thymidylate synthase that was unfolded and refolded as a control. We have also formed the V316Am-WT heterodimer and report that this heterodimeric enzyme is also active, has a kcat value that is approximately half of that of the wild-type thymidylate synthase dimer, and binds substrate and cofactor with Km values similar to those of the wild-type enzyme.

A C-terminal conformational equilibrium in thymidylate synthase observed by electron paramagnetic resonance spectroscopy.

A spin-label was attached to the C-terminal side chain of Lactobacillus casei thymidylate synthase (TS, EC2.1.1.45), and EPR spectroscopy was used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate analog CB3717. The C244T/V316C mutant TS has only two cysteines, the active site Cys-198 and an engineered cysteine which replaces valine as the C-terminal residue. dUMP was used to block the active-site cysteine while the C-terminus was reacted with the spin-label 4-maleimido-2,2,6,6- tetramethylpiperidinyl-1-oxy. Exclusive attachment of the label to the C-terminal cysteine was verified by a study of the labeled enzyme's reaction with 5,5'-dithiobis(2-nitrobenzoic acid). EPR spectra of the labeled enzyme and its complexes were composed of two components corresponding to populations of both flexible and more immobilized forms of the C-terminus (tau C = 1 and 9.7 ns, respectively). Ligand binding increased the population of the more immobilized form of the C-terminus with the following series: free enzyme < E.dUMP approximately dTMP approximately E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced perturbation of the conformational equilibrium was titratable and indicated approximate Kd values of 3 and 13 microM for formation of the E.dUMP and E.CB3717 binary complexes, respectively, and 7 microM for the binding of CB3717 to the E.dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal residue in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the position of the equilibrium changes in the presence of ligands.

Interaction of thymidylate synthase with pyridoxal 5'-phosphate as studied by UV/visible difference spectroscopy and molecular modeling.

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillus casei thymidylate synthase (TS), competitive with respect to the nucleotide substrate dUMP (Chen et al., 1989). The UV/vis difference spectra of TS-PLP complexes show lambda max at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, and lambda min at 388 nm due to depletion of free PLP. At high concentrations of PLP a new absorbance at 430 nm forms due to nonspecific Schiff base formation between PLP and lysine residues of the enzyme. Using spectral titration at 328 nm, the binding constant of the specific TS-PLP complex was determined to be 0.5 microM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP complex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules which bind to the active site of TS. Analogs of PLP which do not contain the phosphate or the aldehyde moieties of PLP bound poorly to the enzyme, thus demonstrating the importance of these functional groups for binding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as the wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of 2'-deoxyuridine 5'-monophosphate and 5,10-methylenetetrahydrofolate.

Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 A). This movement from an "open" unliganded state to the "closed" complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. The 2.7-A binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20 degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-A crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex structures open new venues for the design of novel TS inhibitors.

Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme.

The C-terminal residue of thymidylate synthase (TS) is highly conserved and has been implicated in cofactor binding, catalysis, and a conformational change. The codon for the C-terminal valine of Lactobacillus casei TS has been replaced with those for 19 other amino acids and the amber stop codon. Fourteen of the resulting mutant proteins were active by genetic complementation using a Thy- strain of Escherichia coli, and 18 mutants were active by in vitro assay. Only the aspartate and amber mutations had undetectable activity. All of the mutants were expressed at high levels (5-30% of soluble protein) and were purified by phosphocellulose chromatography. In general, the alterations at position 316 led to little effect on the Km for dUMP, an increase in Km for the folate cofactor, and a decrease in kcat. The observations show that TS can tolerate the substitution of most amino acids for valine at the C-terminus without a complete loss of activity, that hydrophobic substitutions are preferred, and that the C-terminal side chain is involved in both cofactor binding and catalysis. There was an excellent correlation between log kcat and hydrophobicity of the side chain at position 316 and an inverse correlation between log Km and the hydrophobicity of this residue. Kinetic parameters of the cofactor-independent TS-catalyzed dehalogenation of BrdUMP showed no variation with the side chain at position 316. In context of the structure of TS, it is proposed that binding of the cofactor triggers a conformational change in which the C-terminal side chain undergoes hydrophobic interactions that stabilize a rate-limiting transition state of the TS reaction.

Thymidylate synthase with a C-terminal deletion catalyzes partial reactions but is unable to catalyze thymidylate formation.

The V316Am mutant of Lactobacillus casei thymidylate synthase has a single amino acid deletion at the C-terminus which abolishes catalysis of dTMP formation. However, V316Am catalyzes two partial reactions which require covalent catalysis: a CH2H4folate-dependent exchange of the 5-hydrogen of dUMP for protons in water and a thiol-dependent dehalogenation of 5-bromo- and 5-iodo-dUMP. These reactions proceed with kcat and Km values similar to those of the wild-type TS-catalyzed reactions. dUMP, dTMP, and FdUMP are competitive inhibitors of the debromination reaction with Ki values similar to those obtained with wild-type enzyme. These results show that removal of the terminal valine does not alter the ability of the enzyme to bind to or form covalent bonds with nucleotide ligands. V316Am also forms a covalent ternary complex with FdUMP and CH2H4folate. However, the affinity of the TS-FdUMP complex for the cofactor is reduced, and the rate of covalent ternary complex formation and its stability are significantly lower than with wild-type TS. These results allow us to place the major defects of the mutation on steps that occur subsequent to initial CH2H4folate binding.