The crystal structure of a bacterial, bifunctional 5,10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase.

The structure of a bifunctional 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase from Escherichia coli has been determined at 2.5 A resolution in the absence of bound substrates and compared to the NADP-bound structure of the homologous enzyme domains from a trifunctional human synthetase enzyme. Superposition of these structures allows the identification of a highly conserved cluster of basic residues that are appropriately positioned to serve as a binding site for the poly-gamma-glutamyl tail of the tetrahydrofolate substrate. Modeling studies and molecular dynamic simulations of bound methylene-tetrahydrofolate and NADP shows that this binding site would allow interaction of the nicotinamide and pterin rings in the dehydrogenase active site. Comparison of these enzymes also indicates differences between their active sites that might allow the development of inhibitors specific to the bacterial target.

Purification, crystallization, and preliminary x-ray studies of a bifunctional 5,10-methenyl/methylene-tetrahydrofolate cyclohydrolase/dehydrogenase from Escherichia coli.

A bifunctional enzyme that catalyzes the conversion of formyltetrahydrofolate to methylene-tetrahydrofolate (5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylene tetrahydrofolate dehydrogenase), has been subcloned from a cDNA library, purified to homogeneity, and crystallized. The crystals belong to space group I222, with unit cell dimensions of a = 64.5 A, b = 84.9 A, c = 146.1 A. The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme monomer, and a specific volume of the unit cell of 3.2 A3/Da. The crystals diffract to at least 2.8 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. A 2.56 A resolution native data set has been collected at beamline X12-C at the NSLS.

Synthesis, kinetics, and structural studies of a photolabile caged isocitrate: a catalytic trigger for isocitrate dehydrogenase.

A biologically inactive photolabile derivative of isocitrate has been synthesized and characterized. The caged isocitrate is photolyzed to isocitrate with a rate constant of 234 s-1, a half-life of 3 ms, and a quantum yield of 0.3 at pH = 6.4. Caged isocitrate (1-(2-nitrophenyl)ethyl 1-hydroxy-1,2-dicarboxy-3- propanecarboxylate) was synthesized in a straightforward synthetic manner starting with racemic isocitric acid lactone. Laser pulse photolysis at a wavelength of 355 nm was used to determine the rate of photolysis and the quantum yield and to quantify the amount of energy needed for quantitative conversion of the caged isocitrate to free isocitrate. Enzymatic conversion of the liberated isocitrate to alpha-ketoglutarate was achieved in solution as well as in wild-type and mutant isocitrate dehydrogenase (IDH) protein crystals. The X-ray crystal structures of wild-type IDH soaked with photolabile caged isocitrate and Mg2+ and void of nicotinamide adenine dinucleotide phosphate were solved at 2.5 A resolution before and after photolysis and compared by difference mapping against previously determined enzyme structures. Prior to photolysis the enzyme active site contains a low occupancy of bound free Mg2+ in the metal binding site but no observable bound isocitrate, whereas after photolysis the enzyme is complexed to liberated isocitrate and Mg2+ with binding interactions identical to those of previously determined substrate complexes. Single-crystal spectroscopy of the crystals after flash photolysis in the presence of substrates shows production of bound enzyme-substrate complexes and reduced nicotinamide adenine dinucleotide phosphate induced by the photolytic event.

Mutagenesis and Laue structures of enzyme intermediates: isocitrate dehydrogenase.

Site-directed mutagenesis and Laue diffraction data to 2.5 A resolution were used to solve the structures of two sequential intermediates formed during the catalytic actions of isocitrate dehydrogenase. Both intermediates are distinct from the enzyme-substrate and enzyme-product complexes. Mutation of key catalytic residues changed the rate determining steps so that protein and substrate intermediates within the overall reaction pathway could be visualized.

Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP(+)-dependent isocitrate dehydrogenase from Escherichia coli.

Two site-directed mutants of isocitrate dehydrogenase (IDH) of Escherichia coli have been studied by site-directed mutagenesis kinetic and structural studies. Substitution of phenylalanine for tyrosine at position 160 (Y160F) showed 0.4% of the kcat of wild-type with isocitrate as substrate, while the Km for isocitrate remained unchanged. When the postulated intermediate, oxalosuccinate, was enzymatically decarboxylated, Y160F showed a higher kcat and a similar Km to the wild type values. The rate of reduction of oxalosuccinate to isocitrate by the Y160F mutant was greatly decreased relative to the wild-type. Substitution of methionine for lysine at position 230 decreased kcat to 1.1% of that of the wild-type and Km increased by a factor of 500-600. The decarboxylation of oxalosuccinate was undetectable for the K230M mutant. The structure of the site-directed mutants of IDH with and without a bound complex of isocitrate and Mg2+ was solved at 2.5 A resolution and compared by difference mapping against previously determined enzyme structures. The structural studies show that (i) the overall protein-folding side chain conformations and active sites of both mutants are isomorphous with wild-type enzyme, (ii) isocitrate and magnesium bind to both enzyme mutants with the same relative conformation and binding interactions as wild-type enzyme, and (iii) the mutated side chains (Phe 160 and Met 230) are positioned for catalysis in a similar conformation as that observed for the wild-type enzyme. Hence, the alteration of the side chain functional groups is directly related to the loss of enzyme activity. Possible roles of the active site tyrosine and lysine are discussed.

Preliminary crystallographic analysis of methane mono-oxygenase hydroxylase from Methylosinus trichosporium OB3b.

The hydroxylase component of the enzyme methane mono-oxygenase from Methylosinus trichosporium OB3b has been crystallized in the orthorhombic space group C222(1) with unit cell dimensions a = 264.5 A, b = 71.2 A, c = 139.4 A. The crystals grow as square, thick plates and diffract to beyond 2 A resolution. There is one half of the hydroxylase dimer in the asymmetric unit.