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J N Jansonius

Publications and source records attributed to J N Jansonius.

At least 55 records · Page 3Linked to original sources
PubMed1994

Crystallization and preliminary X-ray analysis of recombinant 1-aminocyclopropane-1-carboxylate synthase from apple. A key enzyme in the biosynthesis of the plant hormone ethylene.

Crystals of recombinant 1-aminocyclopropane-1-carboxylate synthase from apple have been obtained with polyethylene glycol as precipitant using a combination of vapour diffusion and macroseeding techniques. The crystals are of space group P2(1), with unit-cell constants a = 53.7 A, b = 69.3 A, c = 123.7 A and beta = 89.9 degrees. The asymmetric unit content is a 1-aminocyclopropane-1-carboxylate synthase dimer with a molecular mass of 94 kDa. Diffraction extends to 2.2 A resolution.

Crystallization↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies of recombinant human ornithine aminotransferase.

Human liver ornithine aminotransferase was expressed in Escherichia coli and purified by ammonium sulfate fractionation and anion exchange column chromatography. The purified recombinant enzyme is fully active and crystallized readily over a wide range of polyethylene glycol concentrations. The crystals belong to the trigonal space group P3(1)21 (or its enantiomorph P3(2)21) with unit cell parameters a = b = 116.3 A, and c = 190.0 A, alpha = beta = 90 degrees, gamma = 120 degrees. There are three monomers per asymmetric unit. Self-rotation function studies revealed both 2-fold and 3-fold non-crystallographic symmetry, with the local 3-fold axis being tilted 15 degrees from the c axis and perpendicular to a crystallographic dyad. A complete native data set to 2.3 A resolution was collected using synchrotron radiation.

Cloning, Molecular↗
PubMed1994

Crystallization and preliminary X-ray analysis of wild-type and K272A mutant glutamate 1-semialdehyde aminotransferase from Synechococcus.

Crystals of the pyridoxal-5'-phosphate dependent enzyme glutamate-1-semialdehyde aminotransferase (EC 5.4.3.8) from Synechococcus have been grown from polyethylene glycol solutions. The wild-type enzyme crystallizes in space group P2(1)2(1)2(1), with cell dimensions a = 68.4 A, b = 108.0 A, c = 122.6 A. The inactive mutant in which the cofactor-binding lysine 272 residue is replaced by alanine (K272A) gives monoclinic crystals of space group P2(1) with cell dimensions a = 67.1 A, b = 108.6 A, c = 124.5 A and beta = 115.7 degrees. These crystal forms diffract to 2.4 A and 2.7 A resolution, respectively.

Crystallization↗
PubMed1994

Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms.

Three crystal structures of wild type E. coli aspartate aminotransferase (E.C.2.6.1.1) in space group P2(1) have been determined at resolution limits between 2.6 and 2.35 A. The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations. The unit cell parameters of the unliganded and the inhibited enzyme are a = 87.2, b = 79.9, c = 89.8 A and beta = 119.1 degrees, and a = 85.4, b = 79.8, c = 89.5 A and beta = 118.6 degrees, respectively. The crystallographic symmetry is pseudo-C222(1). The liganded enzyme structures were solved by difference Fourier techniques from that of a Val39-->Leu mutant partially refined to an R-factor of 0.22 at 2.85 A. They have a "closed" conformation like the chicken mAATase:maleate complex. The models were refined to R-factors of 0.19 (maleate complex) and 0.18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods. The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0.19 at 2.5 A resolution. The structure is in a "half-open" conformation, with the small domain rotated about 6 degrees from the closed conformation. The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase. Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase. The C alpha-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for alpha-deprotonation of the substrate L-aspartate. Comparisons with earlier determined eAATase structures in space group C222(1) revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies. The present P2(1) structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes. They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme.

Amino Acid Sequence↗
PubMed1994

Crystalline mitochondrial aspartate aminotransferase exists in only two conformations.

The subunits of the alpha 2-dimeric enzyme aspartate aminotransferase are composed of two distinct domains, one large and one small. The active sites are situated close to both the intersubunit and the interdomain interface. Binding of substrate analogues to the active site induces a large conformational change in the enzyme, whereby the small domain rotates by 13 degrees relative to the large domain and completely buries the ligand. We have determined the crystal structures of chicken mitochondrial aspartate aminotransferase (mAATase) in two new crystal forms. A comparison of the structures of mAATase in five crystal forms, including both the unliganded and the liganded enzyme, shows that mAATase exists in either one of two unique conformations, with only minimal adaptations to the crystal lattice. This suggests that both the open, unliganded and closed, liganded structure of mAATase are, to a large extent, stabilized by intramolecular interactions, and are consequently representative of functional states of the protein in solution. A 2-fold-symmetric packing interaction between small domains occurring identically in three crystal forms of mAATase is described.

Animals↗
PubMed1994

Significant improvement to the catalytic properties of aspartate aminotransferase: role of hydrophobic and charged residues in the substrate binding pocket.

The substrate specificity of tyrosine aminotransferase (eTAT) from Escherichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspartate aminotransferase (eAAT). These residues are not directly involved in the catalytic process. The single mutant eAAT V39L possesses greater values of kcat/KM not only for tyrosine but also for aspartate and glutamate. In contrast, the double mutant eAAT P141E,A293R and also the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of kcat/KM. The converse mutants of tyrosine aminotransferase, in which critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, Val37) were transferred into equivalent positions of eTAT, exhibited generally decreased values of kcat/KM for both dicarboxylic and aromatic substrates. On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate that the different substrate specificities of eAAT and eTAT are due to multiple side chain differences and minor rearrangements of the backbone. The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated closure of the active site upon substrate binding.

Amino Acid Sequence↗
PubMed1994

Three-dimensional structure of a mutant E. coli aspartate aminotransferase with increased enzymic activity.

The aspartate and tyrosine aminotransferases from Escherichia coli have 43% sequence identity and nearly identical active sites. Both are equally good enzymes for dicarboxylate substrates, but the latter transaminates aromatic amino acids 1000 times faster. In an attempt to discover the critical residues for this differential substrate specificity, the aspartate aminotransferase mutant V39L has recently been prepared. It showed improved Kcat/Km values for aspartate, glutamate and tyrosine and the corresponding oxo acids, mainly due to two to ten times lower Km values. For example, the Km values of V39L (wild type) for Asp and Glu are 0.12 (1.0) and 0.85 (2.7) mM respectively. The mutant was co-crystallized with 30 mM maleate from both polyethylene glycol and ammonium sulfate. Both structures were solved and refined to R-factors of 0.22 and 0.20 at 2.85 and 2.5 A resolution respectively. They bear strong resemblance to the closed structure of the wild type enzyme complexed with maleate. The unexpected feature is that, for the first time, the closed form was produced in crystals grown from ammonium sulfate. It is concluded that the mutation has shifted the conformational equilibrium towards the closed form, which leads to generally reduced substrate Kms.

Aspartate Aminotransferases↗
PubMed1993

Crystal structures of true enzymatic reaction intermediates: aspartate and glutamate ketimines in aspartate aminotransferase.

The crystal structures of the stable, closed complexes of chicken mitochondrial aspartate aminotransferase with the natural substrates L-aspartate and L-glutamate have been solved and refined at 2.4- and 2.3-A resolution, respectively. In both cases, clear electron density at the substrate-coenzyme binding site unequivocally indicates the presence of a covalent intermediate. The crystallographically identical environments of the two subunits of the alpha 2 dimer allow a simple, direct correlation of the coenzyme absorption spectra of the crystalline enzyme with the diffraction results. Deconvolution of the spectra of the crystalline complexes using lognormal curves indicates that the ketimine intermediates constitute 76% and 83% of the total enzyme populations with L-aspartate and L-glutamate, respectively. The electron density maps accommodate the ketimine structures best in agreement with the independent spectral data. Crystalline enzyme has a much higher affinity for keto acid substrates compared to enzyme in solution. The increased affinity is interpreted in terms of a perturbation of the open/closed conformational equilibrium by the crystal lattice, with the closed form having greater affinity for substrate. The crystal lattice contacts provide energy required for domain closure normally supplied by the excess binding energy of the substrate. In solution, enzyme saturated with amino/keto acid substrate pairs has a greater total fraction of intermediates in the aldehyde oxidation state compared to crystalline enzyme. Assuming the only difference between the solution and crystalline enzymes is in conformational freedom, this difference suggests that one or more substantially populated, aldehydic intermediates in solution exist in the open conformation. Quantitative analyses of the spectra indicate that the value of the equilibrium constant for the open-closed conformational transition of the liganded, aldehydic enzyme in solution is near 1. The C4' pro-S proton in the ketimine models is oriented nearly perpendicularly to the plane of the pyridine ring, suggesting that the enzyme facilitates its removal by maximizing sigma-pi orbital overlap. The absence of a localized water molecule near Lys258 dictates that ketimine hydrolysis occurs via a transiently bound water molecule or from an alternative, possibly more open, structure in which water is appropriately bound. A prominent mechanistic role for flexibility of the Lys258 side chain is suggested by the absence of hydrogen bonds to the amino group in the aspartate structure and the relatively high temperature factors for these atoms in both structures.

Animals↗
PubMed1993

Dialkylglycine decarboxylase structure: bifunctional active site and alkali metal sites.

The structure of the bifunctional, pyridoxal phosphate-dependent enzyme dialkylglycine decarboxylase was determined to 2.1-angstrom resolution. Model building suggests that a single cleavage site catalyzes both decarboxylation and transamination by maximizing stereoelectronic advantages and providing electrostatic and general base catalysis. The enzyme contains two binding sites for alkali metal ions. One is located near the active site and accounts for the dependence of activity on potassium ions. The other is located at the carboxyl terminus of an alpha helix. These sites help show how proteins can specifically bind alkali metals and how these ions can exert functional effects.

Amination↗
PubMed1993

Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue. Structural and catalytic properties.

If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence [Ziak, M., Jaussi, R., Gehring, H. and Christen, P. (1990) Eur. J. Biochem. 187, 329-333]. The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution. The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site. If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme [lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm], the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated. Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds. The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme. The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5. Reconstitution of the mutant apoenzyme with [4'-3H]pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme. Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue. The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule. Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

Animals↗
PubMed1993

X-ray structure of a decameric cyclophilin-cyclosporin crystal complex.

Human cyclophilin A (CypA), a ubiquitous intracellular protein of 165 amino acids, is the major receptor for the cyclic undecapeptide immunosuppressant drug cyclosporin A (CsA), which prevents allograft rejection after transplant surgery and is efficacious in the field of autoimmune diseases. CsA prevents T-cell proliferation by blocking the calcium-activated pathway leading to interleukin-2 transcription. Besides their ability to bind CsA, the cyclophilin isoforms also have peptidyl-prolyl isomerase activity and enhance the rate of protein folding. The macrolide FK506 acts similarly to CsA and its cognate receptor FKBP also has peptidyl-prolyl isomerase activity. Inhibition of this enzymatic activity alone is not sufficient to achieve immunosuppression. A direct molecular interaction between the drug-immunophilin complex (CsA-CypA, or FK506-FKBP) and the phosphatase calcineurin, is responsible for modulating the T-cell receptor signal transduction pathway. Here we describe the crystal structure of a decameric CypA-CsA complex. The crystallographic asymmetric unit is composed of a pentamer of 1:1 cyclophilin-cyclosporin complexes of rather exact non-crystallographic fivefold symmetry. The 2.8 A electron density map is of high quality. The five independent cyclosporin molecules are clearly identifiable, providing an unambiguous picture of the detailed interactions between a peptide drug and its receptor. It broadly confirms the results of previous NMR, X-ray and modelling studies, but provides further important structural details which will be of use in the design of drugs that are analogues of CsA.

Amino Acid Isomerases↗
PubMed1992

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.

Amino Acid Sequence↗
PubMed1992

Domain closure in mitochondrial aspartate aminotransferase.

The subunits of the dimeric enzyme aspartate aminotransferase have two domains: one large and one small. The active site lies in a cavity that is close to both the subunit interface and the interface between the two domains. On binding the substrate the domains close together. This closure completely buries the substrate in the active site and moves two arginine side-chains so they form salt bridges with carboxylate groups of the substrate. The salt bridges hold the substrate close to the pyridoxal 5'-phosphate cofactor and in the right position and orientation for the catalysis of the transamination reaction. We describe here the structural changes that produce the domain movements and the closure of the active site. Structural changes occur at the interface between the domains and within the small domain itself. On closure, the core of the small domain rotates by 13 degrees relative to the large domain. Two other regions of the small domain, which form part of the active site, move somewhat differently. A loop, residues 39 to 49, above the active site moves about 1 A less than the core of the small domain. A helix within the small domain forms the "door" of the active site. It moves with the core of the small domain and, in addition, shifts by 1.2 A, rotates by 10 degrees, and switches its first turn from the alpha to the 3(10) conformation. This results in the helix closing the active site. The domain movements are produced by a co-ordinated series of small changes. Within one subunit the polypeptide chain passes twice between the large and small domains. One link involves a peptide in an extended conformation. The second link is in the middle of a long helix that spans both domains. At the interface this helix is kinked and, on closure, the angle of the kink changes to accommodate the movement of the small domain. The interface between the domains is formed by 15 residues in the large domain packing against 12 residues in the small domain and the manner in which these residues pack is essentially the same in the open and closed structures. Domain movements involve changes in the main-chain and side-chain torsion angles in the residues on both sides of the interface. Most of these changes are small; only a few side-chains switch to new conformations.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Crystal structures explain functional properties of two E. coli porins.

Porins form aqueous channels that aid the diffusion of small hydrophilic molecules across the outer membrane of Gram-negative bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel beta-barrel containing a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the molecular level functional characteristics and their alterations by known mutations.

Amino Acid Sequence↗
PubMed1992

The structure of neutral protease from Bacillus cereus at 0.2-nm resolution.

The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution. The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity. The active-site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.

Amino Acid Sequence↗
PubMed1992

X-ray structure refinement and comparison of three forms of mitochondrial aspartate aminotransferase.

The X-ray crystal structures of three forms of the enzyme aspartate aminotransferase (EC 2.6.1.1) from chicken heart mitochondria have been refined by least-squares methods: holoenzyme with the co-factor pyridoxal-5'-phosphate bound at pH 7.5 (1.9 A resolution), holoenzyme with pyridoxal-5'-phosphate bound at pH 5.1 (2.3 A resolution) and holoenzyme with the co-factor pyridoxamine-5'-phosphate bound at pH 7.5 (2.2 A resolution). The crystallographic agreement factors [formula: see text] for the structures are 0.166, 0.130 and 0.131, respectively, for all data in the resolution range from 10.0 A to the limit of diffraction for each structure. The secondary, super-secondary and domain structures of the pyridoxal-phosphate holoenzyme at pH 7.5 are described in detail. The surface area of the interface between the monomer subunits of this dimeric alpha 2 protein is unusually large, indicating a very stable dimer. This is consistent with biochemical data. Both subunit and domain interfaces are relatively smooth compared with other proteins. The interactions of the protein with its co-factor are described and compared among the three structures. Observed changes in co-factor conformation may be related to spectral changes and the energetics of the catalytic reaction. Small but significant adjustments of the protein to changes in co-factor conformation are seen. These adjustments may be accommodated by small rigid-body shifts of secondary structural elements, and by packing defects in the protein core.

Aspartate Aminotransferases↗
PubMed1992

Crystallization and preliminary X-ray analysis of pig kidney DOPA decarboxylase.

DOPA decarboxylase from pig kidney, an alpha 2 dimeric enzyme of Mr = 107,000, has been crystallized by the vapour diffusion method with ammonium sulphate as precipitant. The crystals belong to the space group P6(2) (or its enantiomer P6(4)) and have unit cell dimensions of a = b = 155.9 A, c = 87.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. They diffract to 2.6 A resolution. There is one dimeric molecule per asymmetric unit. Rotation function studies have revealed the orientation of the non-crystallographic 2-fold axis of the dimer in the asymmetric unit.

Animals↗