Three-dimensional structure of aspartate aminotransferase from Escherichia coli at 2.8 A resolution

The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.     

Crystallization and preliminary X-ray studies of an aspartate aminotransferase mutant from Escherichia coli

Mutant aspartate aminotransferase V39L (Val39 replaced by Leu) from Escherichia coli has been crystallized into a monoclinic cell from a polyethylene glycol solution (pH 7.5) by vapor diffusion. The space group and the unit cell dimensions have been determined using a precession camera, a CAD4 diffractometer and a Nicolet Xentronics area detector to be P2(1) with a = 86.8 A, b = 79.9 A, c = 89.4 A, beta = 118.74 degrees. The crystals diffract to better than 2.3 A and are suitable for X-ray structure analysis.     

2.8-A-resolution crystal structure of an active-site mutant of aspartate aminotransferase from Escherichia coli

The three-dimensional structure of a mutant of the aspartate aminotransferase from Escherichia coli, in which the active-site lysine has been substituted by alanine (K258A), has been determined at 2.8-A resolution by X-ray diffraction. The mutant enzyme contains pyridoxamine phosphate as cofactor. The structure is compared to that of the mitochondrial aspartate aminotransferase. The most striking differences, aside from the absence of the lysine side chain, occur in the positions of the pyridoxamine group and of tryptophan 140.     

Preliminary X-ray data for aspartate aminotransferase from Escherichia coli

Crystals of the aspartate aminotransferase from Escherichia coli (aspC gene product) have been examined by X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2221. Unit cell dimensions are a = 156 A, b = 87.6 A, c = 80.6 A and alpha = beta = gamma = 90 degrees. There is one protein subunit of molecular weight 43,600 per asymmetric unit.     

Three-dimensional structure of thymidine phosphorylase from Escherichia coli at 2.8 A resolution

The three-dimensional structure of thymidine phosphorylase from Escherichia coli has been determined at 2.8 A resolution using multiple-isomorphous-replacement techniques. The amino acid sequence deduced from the deoA DNA sequence is also reported. Thymidine phosphorylase exists in the crystal as an S-shaped dimer in which the subunits are related by a crystallographic 2-fold axis. Each subunit is composed of a small alpha-helical domain of six helices and a large alpha/beta domain. The alpha/beta domain includes a six-stranded mixed beta-sheet and a four-stranded antiparallel beta-sheet. The active site has been identified by difference Fourier analyses of the binding of thymine and thymidine and lies in a cavity between the small and large domains. The central beta-sheet is splayed open to accommodate a putative phosphate-binding site which is probably occupied by a sulfate ion in the crystal.     

Overproduction and preliminary X-ray characterization of aspartate aminotransferase from Escherichia coli

The aspartate aminotransferase of Escherichia coli was overproduced in cells after genetic manipulation, and was crystallized from a polyethylene glycol solution, pH 7.0. The crystals obtained were of good quality and had diffractions extending beyond 2.4 A. The space group and unit cell dimensions were determined with a precession camera and a four-circle diffractometer to be C222(1), and a = 157.1 A, b = 85.5 A, and c = 79.7 A, respectively. Only one protein subunit is contained in an asymmetric unit.     

The purification and properties of the aspartate aminotransferase and aromatic-amino-acid aminotransferase from Escherichia coli.

A simple and convenient procedure is described for the isolation in good yield of two amino-transferases from various strains of Escherichia coli. On the basis of their substrate specificities one of the enzymes has been classified as an aromatic amino acid aminotransferase and the other as an aspartate aminotransferase, but both act on a wide range of substrates. Pyridoxal phosphate is bound more strongly to the aspartate aminotransferase than to the aromatic amino transferase which cannot be fully re-activated after removal of the prosthetic group. Both enzymes are composed of two subunits which appear to be identical.     

The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.     

Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli.

The Trp phosphorescence spectrum, intensity and decay kinetics of apo-aspartate aminotransferase, pyridoxamine-5P-aspartate-aminotransferase and pyridoxal-5P-aspartate aminotransferase were measured over a temperature range 160-273 K. The fine structure of the phosphorescence spectra in low-temperature glasses, with 0-0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine-5P-enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal-5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0-0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp-sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer. All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal-5P enzyme shows a decrease in lifetime-normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal-5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.     

Structural studies on aspartate aminotransferase from Escherichia coli. Covalent structure

The amino acid sequence of aspartate aminotransferase from Escherichia coli was established by sequence analysis and alignment of 39 tryptic peptides and 7 cyanogen bromide peptides. The total number of amino acid residues of the subunit was 396, and the molecular weight was calculated to be 43,573. A comparison of the primary structure of the E. coli enzyme with all known sequences of the two types of isoenzyme (mitochondrial and cytosolic enzymes) in vertebrates revealed that approximately 25% of all residues are invariant. The amino acid residues which were proposed from crystallographic studies on the vertebrate enzymes to be essential for the enzymic action are well conserved in the E. coli enzyme. The E. coli enzyme shows a similar degree of sequence homology to both the mitochondrial and cytosolic isoenzymes (close to 40%). The finding that the positions of deletions introduced into the sequence of E. coli enzyme to give the maximum homology agree well with those of the mitochondrial enzymes supports the endosymbiotic hypothesis of mitochondrial origin.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate

Phosphoserine aminotransferase (PSAT; EC 2.6.1.52), a member of subgroup IV of the aminotransferases, catalyses the conversion of 3-phosphohydroxypyruvate to l-phosphoserine. The crystal structure of PSAT from Escherichia coli has been solved in space group P212121 using MIRAS phases in combination with density modification and was refined to an R-factor of 17.5% (Rfree=20.1 %) at 2.3 A resolution. In addition, the structure of PSAT in complex with alpha-methyl-l-glutamate (AMG) has been refined to an R-factor of 18.5% (Rfree=25.1%) at 2.8 A resolution. Each subunit (361 residues) of the PSAT homodimer is composed of a large pyridoxal-5'-phosphate binding domain (residues 16-268), consisting of a seven-stranded mainly parallel beta-sheet, two additional beta-strands and seven alpha-helices, and a small C-terminal domain, which incorporates a five-stranded beta-sheet and two alpha-helices. A three-dimensional structural comparison to four other vitamin B6-dependent enzymes reveals that three alpha-helices of the large domain, as well as an N-terminal domain (subgroup II) or subdomain (subgroup I) are absent in PSAT. Its only 15 N-terminal residues form a single beta-strand, which participates in the beta-sheet of the C-terminal domain. The cofactor is bound through an aldimine linkage to Lys198 in the active site. In the PSAT-AMG complex Ser9 and Arg335 bind the AMG alpha-carboxylate group while His41, Arg42 and His328 are involved in binding the AMG side-chain. Arg77 binds the AMG side-chain indirectly through a solvent molecule and is expected to position itself during catalysis between the PLP phosphate group and the substrate side-chain. Comparison of the active sites of PSAT and aspartate aminotransferase suggests a similar catalytic mechanism, except for the transaldimination step, since in PSAT the Schiff base is protonated. Correlation of the PSAT crystal structure to a published profile sequence analysis of all subgroup IV members allows active site modelling of nifs and the proposal of a likely molecular reaction mechanism.     

The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution

An X-ray crystallographic study at 4.5 Å resolution has been carried out with triclinic crystals of chicken mitochondrial aspartate aminotransferase.In the electron density map, the enzyme is clearly visible as an isologous α₂-dimer (105 Å × 60 Å × 50 Å) in which the subunits are associated about a molecular 2-fold axis. Each subunit of dimensions 70 Å × 50 Å × 40 Å contains at least seven helices, one of which is about 50 Å long.Difference maps have revealed the positions of the pyridoxyl and the phosphate moieties of the coenzyme as well as the general substrate binding area. The active sites are on opposite sides of the dimer, about 30 Å apart and close to the intersubunit boundary, so that probably both subunits contribute to each active site. An isolated chain segment, passing in front of the active site and ending in contact with the neighbouring subunit is interpreted as one of the chain termini.     

The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution.

UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening. Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.     

Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 Å resolution

Native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) can be converted by mild proteolysis into a well-defined monomeric fragment of molecular weight 64,000. This fragment retains full specificity towards methionine and tRNA^Met, and has unimpaired activity in both the activation and aminoacylation reactions.This paper describes the structure of the active fragment, as determined by an X-ray crystallographic study at 2.5 Å resolution using five heavy-atom derivatives. The elongated molecule (90 Å × 52 Å × 44 Å) contains several α-helices, which account for 43% of the residues. Three domains can be distinguished in the structure: (1) a central core beginning at the N-terminus, consisting of a five-stranded parallel pleated sheet with α-helices connecting the β-strands; (2) a second domain with less-ordered structure, inserted between the third and fourth strand of the central sheet; (3) a C-terminal domain, beginning after the fifth parallel strand, very rich in α-helices.These three domains are organized in a biglobular structure; one globule contains the first and the second domain (N-terminal globule), the other the third domain. The two globules, linked together by a single chain, are separated by a large cleft.The most salient feature of the structure is the presence, in the N-terminal domain, of a “nucleotide binding fold” similar to that first observed in dehydrogenases. This makes methionyl-tRNA synthetase, and possibly all aminoacyl-tRNA synthetases, a new member of this family of nucleotide binding proteins possessing the characteristic “Rossmann fold”.     

Expression of a hyperthermophilic aspartate aminotransferase in Escherichia coli.

The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity. A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly. The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus. The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts.     

Asymmetrical synthesis of L-homophenylalanine using engineered Escherichia coli aspartate aminotransferase

Site-directed mutagenesis was performed to change the substrate specificity of Escherichia coli aspartate aminotransferase (AAT). A double mutant, R292E/L18H, with a 12.9-fold increase in the specific activity toward L-lysine and 2-oxo-4-phenylbutanoic acid (OPBA) was identified. E. coli cells expressing this mutant enzyme could convert OPBA to L-homophenylalanine (L-HPA) with 97% yield and more than 99.9% ee using L-lysine as amino donor. The transamination product of L-lysine, 2-keto-6-aminocaproate, was cyclized nonenzymatically to form Delta(1)-piperideine 2-carboxylic acid in the reaction mixture. The low solubility of L-HPA and spontaneous cyclization of 2-keto-6-aminocaproate drove the reaction completely toward L-HPA production. This is the first aminotransferase process using L-lysine as inexpensive amino donor for the L-HPA production to be reported.     

Crystallographic study at 2.5 A resolution of the interaction of methionyl-tRNA synthetase from Escherichia coli with ATP

The crystal structure of the tryptic fragment of the methionyl-tRNA synthetase from Escherichia coli, complexed with ATP, has been refined to a crystallographic R-factor of 0.220, at 2.5 A resolution (for 4433 protein atoms). In the last stages of the refinement, the simulated annealing refinement method was fully applied, contributing to a drastic improvement of the model and the identification of the missing atoms. In the final model, the root-mean-square deviation from ideality for bond distances is 0.021 A and for angle distances is 0.054 A. The position of the zinc ion has been confirmed and is located near the active site. The tryptic fragment is composed of two globular domains. The first domain, from the N terminus to Thr360, contains a nucleotide-binding fold into which two long polypeptides of 101 and 70 residues are inserted. The nucleotide-binding fold is strengthened by the presence of the zinc ion in the vicinity of the active site. The second domain, up to Pro526, is mainly alpha-helical. The C-terminal polypeptide, Phe527 to Lys551, folds back towards the first domain, making a link between the two domains. The heptapeptide 528-534 partly shapes a deep cavity that plunges into the central core of the nucleotide-binding fold, where the ATP molecule is located. The adenine ring, deeply buried in the bottom of the cleft, is blocked between the first helix HA, and the strands A and D of the beta-sheet and makes no polar interaction with the enzyme. The 2' and 3' hydroxyl groups of the ribose, whose conformation is C2' endo, interact with the main-chain carbonyl oxygen atoms of Ile231 and Glu241, respectively. The side-chain nitrogen atom of Lys142 is at hydrogen-bonding distance from the ring oxygen O-4' of the ribose. One of the alpha-phosphate oxygen atoms and one of the gamma-phosphate oxygen atoms interact with the imidazole ring of His21, which is well conserved in many of the known synthetases; this indicates a possible crucial role for this residue in binding ATP. The beta-phosphate group is linked to the main-chain carbonyl oxygen atom of Tyr15 through an intermediate water molecule. The gamma-phosphate group interacts with the carbonyl oxygen atom and the side-chain of Asn17.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pre-steady-state kinetics of Escherichia coli aspartate aminotransferase catalyzed reactions and thermodynamic aspects of its substrate specificity

The four half-transamination reactions [the pyridoxal form of Escherichia coli aspartate aminotransferase (AspAT) with aspartate or glutamate and the pyridoxamine form of the enzyme with oxalacetate or 2-oxoglutarate] were followed in a stopped-flow spectrometer by monitoring the absorbance change at either 333 or 358 nm. The reaction progress curves in all cases gave fits to a monophasic exponential process. Kinetic analyses of these reactions showed that each half-reaction is composed of the following three processes: (1) the rapid binding of an amino acid substrate to the pyridoxal form of the enzyme; (2) the rapid binding of the corresponding keto acid to the pyridoxamine form of the enzyme; (3) the rate-determining interconversion between the two complexes. This mechanism was supported by the findings that the equilibrium constants for half- and overall-transamination reactions and the steady-state kinetic constants (Km and kcat) agreed well with the predicted values on the basis of the above mechanism using pre-steady-state kinetic parameters. The significant primary kinetic isotope effect observed in the reaction with deuterated amino acid suggests that the withdrawal of the alpha-proton of the substrates is rate determining. The pyridoxal form of E. coli AspAT reacted with a variety of amino acids as substrates. The Gibbs free energy difference between the transition state and the unbound state (unbound enzyme plus free substrate), as calculated from the pre-steady-state kinetic parameters, showed a linear relationship with the accessible surface area of amino acid substrate bearing an uncharged side chain.(ABSTRACT TRUNCATED AT 250 WORDS)