S-Adenosylmethionine synthetase (MAT,ATP:L-methionine S-adenosltransferase, EC 2.5.1.6) plays a central metabolic role in all organisms. MAT catalyzes the two-step reaction which synthesizes S-adenosylmethionine (AdoMet), pyrophosphate (PPi), and orthophosphate (Pi) from ATP and L-methionine. AdoMet is the primary methyl group donor in biological systems. The first crystal structure of MAT from Escherichia coli has recently been determined [Takusagawa et al. (1995) J. Biol. Chem. 271, 136-147]. In order to elucidate the active site and possible catalytic reaction mechanism, the MAT structures in the crystals grown with the substrate ATP (and BrATP) and the product PPi have been determined (space group P6(2)22; unit cell a = b = 128.9 Angstroms, c= 139.8 Angstroms, resolution limit 2.8 Angstroms; R O.19; Rfree 0.26). The enzyme consists of four identical subunits; two subunits form a spherical dimer, and pairs of these tightly bound dimers form a tetrameric enzyme. Each dimer has two active sites which are located between the subunits. Each subunit consists of three domains related to each other by a pseudo 3-fold symmetry. The crystal structures showed that the ATP molecules were hydrolyzed to ADP and Pi by the enzyme. Those products were found at the active site along with the essential metal ions (K+ and Mg2+). This rather unexpected finding was first confirmed by the structure of the complex with PPi and later by an HPLC analysis. The enzyme hydrolyzed ATP to ADP and Pi in 72 h under the same conditions as the crystallization of the enzyme. In the active site, the diphosphate moiety of ADP and Pi interacts extensively with amino acid residues from the two subunits of the enzyme, whereas the adenine and ribose moieties have little interaction with the enzyme. The enzyme structure is little changed upon binding ADP. All amino acid residues involved in the active site are found to be conserved in the 14 reported sequences of MAT from a wide range of organisms. Thus the structure determined in this study can be utilized as a model for other members of the MAT family. On the basis of the crystal structures, the catalytic reaction mechanisms of AdoMet formation and hydrolysis of tripolyphosphate are proposed.

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The structure of S-adenosylmethionine synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6.) from Escherichia coli has been determined at 3.0 A resolution by multiple isomorphous replacement using a uranium derivative and the selenomethionine form of the enzyme (SeMAT). The SeMAT data (9 selenomethionine residues out of 383 amino acid residues) have been found to have a sufficient phasing power to determine the structure of the 42,000 molecular weight protein by combining them with the other heavy atom derivative data (multiple isomorphous replacement). The enzyme consists of four identical subunits; two subunits form a spherical tight dimer, and pairs of these dimers form a peanut-shaped tetrameric enzyme. Each pair dimer has two active sites which are located between the subunits. Each subunit consists of three domains that are related to each other by pseudo-3-fold symmetry. The essential divalent (Mg2+/Co2+) and monovalent (K+) metal ions and one of the product, Pi ions, were found in the active site from three separate structures.

We report the first three-dimensional structure of fungus-derived glucose dehydrogenase using flavin adenine dinucleotide (FAD) as the cofactor. This is currently the most advanced and popular enzyme used in glucose sensor strips manufactured for glycemic control by diabetic patients. We prepared recombinant nonglycosylated FAD-dependent glucose dehydrogenase (FADGDH) derived from Aspergillus flavus (AfGDH) and obtained the X-ray structures of the binary complex of enzyme and reduced FAD at a resolution of 1.78 Å and the ternary complex with reduced FAD and D-glucono-1,5-lactone (LGC) at a resolution of 1.57 Å. The overall structure is similar to that of fungal glucose oxidases (GOxs) reported till date. The ternary complex with reduced FAD and LGC revealed the residues recognizing the substrate. His505 and His548 were subjected for site-directed mutagenesis studies, and these two residues were revealed to form the catalytic pair, as those conserved in GOxs. The absence of residues that recognize the sixth hydroxyl group of the glucose of AfGDH, and the presence of significant cavity around the active site may account for this enzyme activity toward xylose. The structural information will contribute to the further engineering of FADGDH for use in more reliable and economical biosensing technology for diabetes management.