Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase.

A recombinant form of the flavoenzyme acyl-CoA oxidase from rat liver has been crystallized by the hanging-drop vapour-diffusion technique using PEG 20 000 as a precipitating agent. The crystals grew as yellow prisms, with unit-cell parameters a = 71.05, b = 87.29, c = 213.05 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.21 A(3) Da(-1). The crystals diffract to a resolution of 2.5 A at beamline BL6A of the Photon Factory. Two heavy-atom derivatives have been identified.

Substrate recognition mechanism of thermophilic dual-substrate enzyme.

Aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ttAspAT), has been believed to be specific for an acidic substrate. However, stepwise introduction of mutations in the active-site residues finally changed its substrate specificity to that of a dual-substrate enzyme. The final mutant, [S15D, T17V, K109S, S292R] ttAspAT, is active toward both acidic and hydrophobic substrates. During the course of stepwise mutation, the activities toward acidic and hydrophobic substrates changed independently. The introduction of a mobile Arg292* residue into ttAspAT was the key step in the change to a "dual-substrate" enzyme. The substrate recognition mechanism of this thermostable "dual-substrate" enzyme was confirmed by X-ray crystallography. This work together with previous studies on various enzymes suggest that this unique "dual-substrate recognition" mechanism is a feature of not only aminotransferases but also other enzymes.

Structures of Escherichia coli histidinol-phosphate aminotransferase and its complexes with histidinol-phosphate and N-(5'-phosphopyridoxyl)-L-glutamate: double substrate recognition of the enzyme.

Histidinol-phosphate aminotransferase (HspAT) is a key enzyme on the histidine biosynthetic pathway. HspAT catalyzes the transfer of the amino group of L-histidinol phosphate (Hsp) to 2-oxoglutarate to form imidazole acetol phosphate (IAP) and glutamate. Thus, HspAT recognizes two kinds of substrates, Hsp and glutamate (double substrate recognition). The crystal structures of native HspAT and its complexes with Hsp and N-(5'-phosphopyridoxyl)-L-glutamate have been solved and refined to R-factors of 19.7, 19.1, and 17.8% at 2.0, 2.2, and 2.3 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. Aspartate aminotransferases (AspATs) from many species were classified into aminotransferase subgroups Ia and Ib. The primary sequence of HspAT is less than 18% identical to those of Escherichia coli AspAT of subgroup Ia and Thermus thermophilus HB8 AspAT of subgroup Ib. The X-ray analysis of HspAT showed that the overall structure is significantly similar to that of AspAT of subgroup Ib rather than subgroup Ia, and the N-terminal region moves close to the active site like that of subgroup Ib AspAT upon binding of Hsp. The folding of the main-chain atoms in the active site is conserved between HspAT and the AspATs, and more than 40% of the active-site residues is also conserved. The eHspAT recognizes both Hsp and glutamate by utilizing essentially the same active-site folding as that of AspAT, conserving the essential residues for transamination reaction, and replacing and relocating some of the active-site residues. The binding sites for the phosphate and the alpha-carboxylate groups of the substrates are roughly located at the same position and those for the imidazole and gamma-carboxylate groups at the different positions. The mechanism for the double substrate recognition observed in eHspAT is in contrast to that in aromatic amino acid aminotransferase, where the recognition site for the side chain of the acidic amino acid is formed at the same position as that for the side chain of aromatic amino acids by large-scale rearrangements of the hydrogen bond networks.

Strain is more important than electrostatic interaction in controlling the pKa of the catalytic group in aspartate aminotransferase.

Systematic single and multiple replacement studies have been applied to Escherichia coli aspartate aminotransferase to probe the electrostatic effect of the two substrate-binding arginine residues, Arg292 and Arg386, and the structural effect of the pyridoxal 5'-phosphate-Asn194-Arg386 hydrogen-bond linkage system (PLP-N-R) on the pK(a) value of the Schiff base formed between pyridoxal 5'-phosphate (PLP) and Lys258. The electrostatic effects of the two arginine residues cannot be assessed by simple mutational studies of the residues. PLP-N-R lowers the pK(a) value of the PLP-Lys258 Schiff base by keeping it in the distorted conformation, which is unfavorable for protonation. Mutation of Arg386 eliminates its hydrogen bond with Asn194 and partially disrupts PLP-N-R, thereby relaxing the strain of the Schiff base. On the other hand, mutation of Arg292, the large domain residue that interacts with the small domain residue Asp15, makes the domain opening easier. Because PLP-N-R lies between the two domains, the domain opening increases the strain of the Schiff base. Therefore, the true electrostatic effects of Arg292 and Arg386 could be derived from mutational analysis of the enzyme in which PLP-N-R had been completely disrupted by the Asn194Ala mutation. Through the analyses, we could dissect the electrostatic and structural effects of the arginine mutations on the Schiff base pK(a). The positive charges of the two arginine residues and the PLP-N-R-mediated strain of the Schiff base lower the Schiff base pK(a) by 0.7 and 1.7, respectively. Thus, the electrostatic effect of the arginine residues is not as strong as has historically been thought, and this finding substantiates our recent finding that the imine-pyridine torsion of the Schiff base is the primary determinant (2.8 unit decrease) of the extremely low pK(a) value of the Schiff base [Hayashi, H., Mizuguchi, H., and Kagamiyama, H. (1998) Biochemistry 37, 15076-15085].

Stereochemistry of the transamination reaction catalyzed by aminodeoxychorismate lyase from Escherichia coli: close relationship between fold type and stereochemistry.

Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38].

Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli.

4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 A resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.

Three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase. Optimization of the oxidative half-reaction through alignment of the product with reduced flavin.

The three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, delta(1)-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The (+)HN=C double bond of DPC overlaps the N(5)-C(4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C(4a), thereby augmenting the electron density at C(4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.

Enantioselective Single-Crystal-to-Single-Crystal Photodimerization of Coumarin and Thiocoumarin in Inclusion Complexes with Chiral Host Compounds.

An intermolecular enantioselective photoreaction by a single-crystal-to-single-crystal transformation has been carried out for the first time, as is evident from X-ray structure analysis and X-ray powder diffractometric studies. This reaction, the dimerization of the title compound to cyclobutane derivative 1 (X=O, S), provides a good example for studying the mechanism of topochemical reactions in the crystal.

Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate.

The three-dimensional structures of pyridoxal 5'-phosphate-type aspartate aminotransferase (AspAT) from Thermus thermophilus HB8 and pyridoxamine 5'-phosphate type one in complex with maleate have been determined by X-ray crystallography at 1.8 and 2.6 A resolution, respectively. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into one arm, one small domain, and one large domain. AspATs from many species were classified into aminotransferase subgroups Ia and Ib. The enzyme belongs to subgroup Ib, its sequence being less than 16% identical to the primary sequences of Escherichia coli, pig cytosolic, and chicken mitochondrial AspATs, which belong to subgroup Ia whose sequences are more than 40% identical and whose three-dimensional structures are quite similar with the active site residues almost completely conserved. The first X-ray analysis of AspAT subgroup Ib indicated that the overall and the active site structures are essentially conserved between the AspATs of subgroup Ia and the enzyme of subgroup Ib, but there are two distinct differences between them. (1) In AspAT subgroup Ia, substrate (or inhibitor) binding induces a large movement of the small domain as a whole to close the active site. However, in the enzyme of subgroup Ib, only the N-terminal region (Lys13-Val30) of the small domain approaches the active site to interact with the maleate. (2) In AspAT subgroup Ia, Arg292 recognizes the side chain carboxylate of the substrate; however, residue 292 of the enzyme in subgroup Ib is not Arg, and in place of Arg292, Lys109 forms a salt bridge with the side chain carboxylate. The thermostability of the enzyme is attained at least in part by the high content of Pro residues in the beta-turns and the marked increase in the number of salt bridges on the molecular surface compared with the mesophilic AspAT.

Structural and mechanistic studies on D-amino acid oxidase x substrate complex: implications of the crystal structure of enzyme x substrate analog complex.

As an extension of our recent X-ray crystallographic determination of the tertiary structure of D-amino acid oxidase (DAO) [Mizutani, H. et al. (1996) J. Biochem. 120, 14-17], we solved the crystal structure of the complex of DAO with a substrate analog, o-aminobenzoate (OAB). The alignment between flavin and OAB in the crystal structure of the complex is consistent with charge-transfer interaction through the overlap between the highest occupied molecular orbital of OAB and the lowest unoccupied molecular orbital of flavin. Starting with the atomic coordinates of this complex as the initial model, we carried out molecular mechanics simulation for the DAO-D-leucine complex and thus obtained a model for the enzyme-substrate complex. According to the enzyme-substrate complex model, the alpha-proton is pointed toward N(5) of flavin while the lone-pair of the substrate amino group can approach C(4a) of flavin within an interacting distance. This model as well as DAO-OAB complex enables the evaluation of the substrate-flavin interaction prior to electron transfer from the substrate to flavin and provides two possible mechanisms for the reductive-half reaction of DAO, i.e., the electron-proton-electron transfer mechanism and the ionic mechanism.

Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

Three-dimensional structure of porcine kidney D-amino acid oxidase at 3.0 A resolution.

The X-ray crystallographic structure of porcine kidney D-amino acid oxidase, which had been expressed in Escherichia coli transformed with a vector containing DAO cDNA, was determined by the isomorphous replacement method for the complex form with benzoate. The known amino acid sequence, FAD and benzoate were fitted to an electron density map of 3.0 A resolution with an R-factor of 21.0%. The overall dimeric structure exhibits an elongated ellipsoidal framework. The prosthetic group, FAD, was found to be in an extended conformation, the isoalloxazine ring being buried in the protein core. The ADP moiety of FAD was located in the typical beta alpha beta dinucleotide binding motif, with the alpha-helix dipole stabilizing the pyrophosphate negative charge. The substrate analog, benzoate, is located on the re-face of the isoalloxazine ring, while the si-face is blocked by hydrophobic residues. The carboxylate group of benzoate is ion-paired with the Arg283 side chain and is within interacting distance with the hydroxy moiety of Tyr228. The phenol ring of Tyr224 is located just above the benzene ring of benzoate, implying the importance of this residue for catalysis. There is no positive charge or alpha-helix dipole near N(1) of flavin. Hydrogen bonds were observed at C(2) = O, N(3)-H, C(4) = O, and N(5) of the flavin ring.

Crystallization of expressed porcine kidney D-amino acid oxidase and preliminary X-ray crystallographic characterization.

The cDNA for porcine kidney D-amino acid oxidase (DAO) was cloned by means of the reverse transcription-polymerase chain reaction system from porcine kidney RNA and over-expressed in Escherichia coli which had been transformed with a vector containing the DAO cDNA. The expressed DAO was purified to homogeneity by a three-step procedure, i.e., heat-treatment, DEAE Sepharose column chromatography, and hydroxyapatite column chromatography. The purified DAO preparation, rDAO (recombinant DAO), showed an identical UV-visible absorption spectrum and catalytic activity with those of the wild-type enzyme purified from porcine kidney. Crystallization of rDAO was performed by the hanging-drop method and crystals of suitable quality for X-ray crystallography were obtained. The crystals so obtained diffracted to 2.5 A with a conventional X-ray source, and to 2.0 A with synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 110.3, b = 92.9, c = 71.6 A. A Vm value of 2.35 A3/Da indicates that there are two subunits related by a twofold non-crystallographic axis in the asymmetric unit. Two heavy atom derivatives have been identified.

NMR studies of 1H resonances in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli.

We have recorded 500-MHz 1H NMR spectra in the 10-18-ppm range for aspartate aminotransferase from Escherichia coli and for three specific mutant forms. Histidine 143 has been replaced by either alanine or asparagine. In the third mutant, tryptophan 140 has been replaced by phenylalanine. The NMR spectrum of the native enzyme is very similar to that of porcine cytosolic aspartate aminotransferase in the most downfield region. However, the resonances of the proton on the ring nitrogen of the pyridoxal 5'-phosphate (peak A) and on the His-143 imidazole ring (peak B) of the E. coli enzyme are broader and more readily lost at low pH or higher temperatures than those of the porcine enzyme. The possible role of tautomerism in promoting such broadening is discussed. In the histidine mutant proteins, peak A of the pyridoxal 5'-phosphate form is too broad to see under most conditions but is clearly present in the pyridoxamine phosphate form. Peak B is missing in the 2 histidine mutants. Observation of nuclear Overhauser effects further confirms the identity of B as the resonance of HN epsilon 2 of His-143 and that of peak D at approximately 11.8 ppm as HN epsilon 2 of His-189. The mutant spectra also provide insight into electronic interactions between groups in and near the active site which confirm and supplement conclusions drawn from spectra of porcine cAspAT. While no clear loss of a peak was observed for the Trp-140 mutant in its free form, the spectrum of the succinate complex lacked a strong band at 11.26 ppm. This may represent the Trp-140 indole NH proton which has been shifted downfield by binding to a succinate carboxylate group. While our results confirm the basic similarity of cytosolic aspartate aminotransferase and E. coli aspartate aminotransferase 1H NMR spectra, they also point out differences that may be useful in identifying resonances. A large number of mutant proteins have been prepared for the E. coli enzyme. The present results provide essential information for future study of these mutants and for study of NMR spectra of isotopically labeled enzyme.

X-ray crystallographic study of pyridoxamine 5'-phosphate-type aspartate aminotransferases from Escherichia coli in three forms.

The three-dimensional structures of pyridoxamine 5'-phosphate-type aspartate aminotransferase from Escherichia coli and its complexes with maleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 A resolution, respectively. The enzyme is a dimeric form comprising two identical subunits, each of which is divided into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the "open" to the "closed" form, resulting in closure of the active site by the small domain movement, as was observed in the pyridoxal 5'-phosphate-type enzyme. In the open form, three hydrophobic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydrophobic by charge compensation and release of water molecules, facilitating the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydrogen bonding network, guaranteeing the stereo-specificity of the catalysis and giving a Michaelis complex model. Contrary to our expectation, the glutarate complex was in the open form, suggesting that the equilibrium between the open and closed forms lies far toward the open form in solution. The water molecules located in the active site pocket were almost completely conserved between Escherichia coli and chicken mitochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.

Role of an active site residue analyzed by combination of mutagenesis and coenzyme analog.

Asp222 of aspartate aminotransferase is an active-site residue which interacts with the pyridine nitrogen of the coenzyme, pyridoxal 5'-phosphate (PLP). The roles of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase have previously been explored by site-directed mutagenesis. These studies confirmed that a negatively charged residue at position 222 is essential for catalysis, but the reason for this remained speculative. In the present studies, the roles of Asp222 were clarified experimentally by analyzing the mutant D222A enzyme (Asp222 replaced by Ala) reconstituted with the coenzyme analog N(1)-methylated PLP (N-MePLP). Spectroscopic and kinetic analyses showed that Asp222 stabilizes the protonated N(1) of PLP, raising the pKa value of N(1) by more than five units, in the active site of AspAT. The positive charge at N(1) accelerates abstraction of the alpha-proton from the amino acid substrate, stabilizing the transition state by 1.4 to 4.5 kcal.mol-1 in the reaction with aspartate. X-ray crystallographic (2.0 A resolution) and CD spectroscopic studies suggest that the coenzyme analog is not held in a proper orientation within the active site of D222A (N-MePLP). This may account for the finding that the catalytic activity was recovered only partially by the reconstitution of D222A with N-MePLP. These results fully support the following postulated role of Asp222: the negative charge of Asp222 stabilizes the positive charge at N(1) of PLP and thereby enhances the function of PLP as an electron sink.