The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

In most organisms, the conversion of ␤-D-galactose to the more metabolically useful glucose 1-phosphate is accomplished by the action of four enzymes that constitute the Leloir pathway (Scheme 1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose by galactose mutarotase. The next step involves the ATP-dependent phosphorylation of ␣-D-galactose by galactokinase to yield galactose 1-phosphate. As indicated in Scheme 1, the third enzyme in the pathway, galactose-1-phosphate uridylyltransferase, catalyzes the transfer of a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDP-galactose is converted to UDP-glucose by UDP-galactose 4-epimerase. In humans, defects in the genes encoding for galactokinase, uridylyltransferase, or epimerase can give rise to the diseased state referred to collectively as galactosemia (1, 2). Although galactosemia is rare, it is potentially lethal with clinical manifestations including intellectual retardation, liver dysfunction, and cataract formation, among others. Indeed, the enzymes of the Leloir pathway have attracted significant research attention for well over 30 -40 years, in part because of their important metabolic role in normal galactose metabolism.As of this year, the three-dimensional structures of all of the enzymes of the Leloir pathway have now been defined. It is thus timely to present in this minireview recent advances in our understanding of the structure and function of these enzymes. For a discussion of the literature prior to 1996, see Ref. 3. Galactose MutarotaseGalactose mutarotase activity was first reported in Escherichia coli in 1965 (4), and the gene encoding it was defined in 1994 (5). Since 1986, genes encoding for proteins with mutarotase activities have been identified in other organisms including Lactococcus lactis (6). With respect to the catalytic mechanism of galactose mutarotase, it was first suggested by Hucho and Wallenfels (7) that the reaction proceeds through the abstraction of the proton from the 1-hydroxyl group of the sugar by an active site base and donation of a proton to the C-5 ring oxygen by an active site acid, thereby leading to ring opening. Subsequent rotation of 180 o about the C-1-C-2 bond followed by abstraction of the proton on the C-5 oxygen and donation of a proton back to the C-1 oxygen generates product. A kinetic analysis of the enzyme from E. coli was recently reported (8).In 2002, the first structure of a galactose mutarotase (from L. lactis) was determined by Thoden et al. (9,10). A ribbon representation of the dimeric enzyme is displayed in Fig. 1. Each subunit contains 339 amino acid residues and adopts a distinctive ␤-sandwich motif. Despite the lack of amino acid sequence homology, the overall topology of the ␤-sandwich is similar to that first observed in domain 5 of ␤-galactosidase from E. coli (11). This ␤-sheet architecture has since been seen in the central domain of copper amine oxidase (12), the C-te...

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution. Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.