Structure determination and refinement at 2.2 angstroms of nitrogenase molybdenum iron-protein from Clostridium pasteurianum

Nitrogenase catalyzes the reduction of dinitrogen through the activity of a complex of two easily separated components known as the MoFe-protein and the Fe-protein. The MoFe-protein is the site of N\sb2 binding and reduction, whereas the Fe-protein provides electrons and binds ATP. MoFe-protein is a large (M\sb{\rm r} = 230,000), \alpha\sb2\beta\sb2, tetrameric enzyme that contains two copies each of two unusual metal clusters known as the FeMo-cofactor and the P-cluster. The crystal structure of MoFe-protein from Clostridium pasteurianum has been determined to 2.2A resolution by a combination of phasing methods including multiwavelength anomalous diffraction, isomorphous replacement, and phase extension involving both solvent flattening and molecular averaging. Both rotating anode and synchrotron (SSRL, CHESS, and Photon Factory) sources were used for diffraction experiments at a variety of wavelengths including 1.0A, 1.54A, 1.74A, and 1.79A. An atomic model of MoFe-protein, with its bound metal-sulfur clusters, 2 Mg\sp{2+} ions, and 1,004 water molecules, has been refined by least-squares techniques against crystallographic data and stereochemical restraints. The refined model has an R-factor of 15.1% based on all measured crystallographic data between 25A and 2.2A. The protein model has root mean-square deviations from ideal bond distances and angles of 0.006A and 1.67\sp\circ, respectively. The crystalloeraphic results are interpreted with respect to the chemical mechanism of N\sb2 reduction and intramolecular electron transfer

Structural Characterization of the Enzyme−Substrate, Enzyme−Intermediate, and Enzyme−Product Complexes of Thiamin Phosphate Synthase

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme−substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures

Structural Characterization of the Enzyme−Substrate, Enzyme−Intermediate, and Enzyme−Product Complexes of Thiamin Phosphate Synthase

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme−substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures