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Gunter Schneider

Publications and source records attributed to Gunter Schneider.

At least 37 records · Page 2Linked to original sources
PubMed2004

Conserved and nonconserved residues in the substrate binding site of 7,8-diaminopelargonic acid synthase from Escherichia coli are essential for catalysis.

The vitamin B(6)-dependent enzyme 7,8-diaminopelargonic acid (DAPA) synthase catalyzes the antepenultimate step in the synthesis of biotin, the transfer of the alpha-amino group of S-adenosyl-l-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA. The Y17F, Y144F, and D147N mutations in the active site were constructed independently. The k(max)/K(m)(app) values for the half-reaction with DAPA of the Y17F and Y144F mutants are reduced by 1300- and 2900-fold, respectively, compared to the WT enzyme. Crystallographic analyses of these mutants do not show significant changes in the structure of the active site. The kinetic deficiencies, together with a structural model of the enzyme-PLP/DAPA Michaelis complex, point to a role of these two residues in recognition of the DAPA/KAPA substrates and in catalysis. The k(max)/K(m)(app) values for the half-reaction with SAM are similar to that of the WT enzyme, showing that the two tyrosine residues are not involved in this half-reaction. Mutations of the conserved Arg253 uniquely affect the SAM kinetics, thus establishing this position as part of the SAM binding site. The D147N mutant is catalytically inactive in both half-reactions. The structure of this mutant exhibits significant changes in the active site, indicating that this residue plays an important structural role. Of the four residues examined, only Tyr144 and Arg253 are strictly conserved in the available amino acid sequences of DAPA synthases. This enzyme thus provides an illustrative example that active site residues essential for catalysis are not necessarily conserved, i.e., that during evolution alternative solutions for efficient catalysis by the same enzyme arose. Decarboxylated SAM [S-adenosyl-(5')-3-methylthiopropylamine] reacts nearly as well as SAM and cannot be eliminated as a putative in vivo amino donor.

Alanine↗
PubMed2003

Crystal structure of aclacinomycin-10-hydroxylase, a S-adenosyl-L-methionine-dependent methyltransferase homolog involved in anthracycline biosynthesis in Streptomyces purpurascens.

Anthracyclines are aromatic polyketide antibiotics, and several of these compounds are widely used as anti-tumor drugs in chemotherapy. Aclacinomycin-10-hydroxylase (RdmB) is one of the tailoring enzymes that modify the polyketide backbone in the biosynthesis of these metabolites. RdmB, a S-adenosyl-L-methionine-dependent methyltransferase homolog, catalyses the hydroxylation of 15-demethoxy-epsilon-rhodomycin to beta-rhodomycin, one step in rhodomycin biosynthesis in Streptomyces purpurascens. The crystal structure of RdmB, determined by multiwavelength anomalous diffraction to 2.1A resolution, reveals that the enzyme subunit has a fold similar to methyltransferases and binds S-adenosyl-L-methionine. The N-terminal domain, which consists almost exclusively of alpha-helices, is involved in dimerization. The C-terminal domain contains a typical alpha/beta nucleotide-binding fold, which binds S-adenosyl-L-methionine, and several of the residues interacting with the cofactor are conserved in O-methyltransferases. Adjacent to the S-adenosyl-L-methionine molecule there is a large cleft extending to the enzyme surface of sufficient size to bind the substrate. Analysis of the putative substrate-binding pocket suggests that there is no enzymatic group in proximity of the substrate 15-demethoxy-epsilon-rhodomycin, which could assist in proton abstraction and thus facilitate methyl transfer. The lack of a suitably positioned catalytic base might thus be one of the features responsible for the inability of the enzyme to act as a methyltransferase.

Amino Acid Sequence↗
PubMed2003

Crystallization and preliminary X-ray diffraction studies of aclacinomycin-10-methyl esterase and aclacinomycin-10-hydroxylase from Streptomyces purpurascens.

Two enzymes participating in the biosynthesis of anthracyclines in Streptomyces purpurascens, aclacinomycin-10-methyl esterase (RdmC) and aclacinomycin-10-hydroxylase (RdmB), have been crystallized. RdmB is a S-adenosyl-methionine-dependent hydroxylase and RdmC hydrolyses the carboxymethyl group of the aglycone skeleton of aclacinomycin. Crystals of RdmB obtained in the presence of S-adenosyl-L-methionine were orthorhombic, space group C222(1), with unit-cell parameters a = 63.2, b = 92.2, c = 115.3 A; diffraction data were collected to 2.1 A resolution. RdmC was crystallized as a complex with the substrate, aclacinomycin T. These crystals diffracted to 1.45 A resolution and belonged to space group P2(1), with unit-cell parameters a = 38.2, b = 84.7, c = 44.3 A, beta = 99.9 degrees.

Anthracyclines↗
PubMed2003

Crystal structure of aclacinomycin methylesterase with bound product analogues: implications for anthracycline recognition and mechanism.

Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 A, respectively. RdmC is built up of two domains. The larger alpha/beta domain shows the common alpha/beta hydrolase fold, whereas the smaller domain is alpha-helical. The active site and substrate binding pocket are located at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.

Aclarubicin↗
PubMed2003

NMR analysis of covalent intermediates in thiamin diphosphate enzymes.

Enzymic catalysis proceeds via intermediates formed in the course of substrate conversion. Here, we directly detect key intermediates in thiamin diphosphate (ThDP)-dependent enzymes during catalysis using (1)H NMR spectroscopy. The quantitative analysis of the relative intermediate concentrations allows the determination of the microscopic rate constants of individual catalytic steps. As demonstrated for pyruvate decarboxylase (PDC), this method, in combination with site-directed mutagenesis, enables the assignment of individual side chains to single steps in catalysis. In PDC, two independent proton relay systems and the stereochemical control of the enzymic environment account for proficient catalysis proceeding via intermediates at carbon 2 of the enzyme-bound cofactor. The application of this method to other ThDP-dependent enzymes provides insight into their specific chemical pathways.

Binding Sites↗
PubMed2003

The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family.

The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy.

Amino Acid Sequence↗
PubMed2003

Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1.

The domestic cat (Felis domesticus) is an important cause of allergic disease worldwide. The major cat allergen 1 (Fel d 1) has been expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Fel d 1 were obtained in 13% 2-methyl-2,4-pentanediol, 0.1 M sodium acetate pH 4.8. The Fel d 1 crystals belong to space group P2(1), with unit-cell parameters a = 43.3, b = 51.5, c = 67.7 A, and diffract to 1.9 A resolution.

Allergens↗
PubMed2003

Genetic alterations in pancreatic carcinoma.

Cancer of the exocrine pancreas represents the fifth leading cause of cancer death in the Western population with an average survival after diagnosis of 3 to 6 months and a five-year survival rate under 5%. Our understanding of the molecular carcinogenesis has improved in the last few years due to the development of novel molecular biological techniques. Pancreatic cancer is a multi-stage process resulting from the accumulation of genetic changes in the somatic DNA of normal cells. In this article we describe major genetic alterations of pancreatic cancer, mutations in the proto-oncogene K-RAS and the tumor suppressors INK4A, TP53 and DPC4/SMAD4. The accumulation of these genetic changes leads to a profound disturbance in cell cycle regulation and continuous growth. The knowledge of the underlying molecular mechanisms will offer new therapeutic and diagnostic options and hopefully improve the outcome of this aggressive disease.

Animals↗
PubMed2003

Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.

Amino Acid Sequence↗
PubMed2002

The dual-specific active site of 7,8-diaminopelargonic acid synthase and the effect of the R391A mutation.

7,8-diaminopelargonic acid (DAPA) synthase (EC 2.6.1.62) is a pyridoxal phosphate (PLP)-dependent transaminase that catalyzes the transfer of the alpha-amino group from S-adenosyl-L-methionine (SAM) to 7-keto-8-aminopelargonic acid (KAPA) to form DAPA in the antepenultimate step in the biosynthesis of biotin. The wild-type enzyme has a steady-state kcat value of 0.013 s(-1), and the K(m) values for SAM and KAPA are 150 and <2 microM, respectively. The k(max) and apparent K(m) values for the half-reaction of the PLP form of the enzyme with SAM are 0.016 s(-1) and 300 microM, respectively, while those for the reaction with DAPA are 0.79 s(-1) and 1 microM. The R391A mutant enzyme exhibits near wild-type kinetic parameters in the reaction with SAM, while the apparent K(m) for DAPA is increased 180-fold. The 2.1 A crystal structure of the R391A mutant enzyme shows that the mutation does not significantly alter the structure. These results indicate that the conserved arginine residue is not required for binding the alpha-amino acid SAM, but it is important for recognition of DAPA.

Alanine↗
PubMed2002

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin.

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 A resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4' carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.

Aminobutyrates↗
PubMed2002

Cyclosporine inhibits growth through the activating transcription factor/cAMP-responsive element-binding protein binding site in the cyclin D1 promoter.

The immunosuppressive agent cyclosporine affects proliferation depending on the cellular system used. In an attempt to study the inhibitory effect of cyclosporine on proliferation of pancreatic acinar cells, we used AR42J cells as a model system. Here we demonstrate that cyclosporine inhibits growth of these cells by inducing G(1) cell cycle arrest. This effect is mediated by the 5' regulatory region of the cyclin D1 gene and leads to a reduction of cyclin D1 mRNA expression and protein abundance. We show that in AR42J cells the proximal cyclin D1 promoter contains a cis-regulated element, which is important for the maintenance of basal transcriptional activity. This element overlaps the described cAMP-responsive element (CRE) and confers cyclosporine sensitivity to the cyclin D1 promoter. Furthermore, the DNA binding activity of the CRE-binding protein (CREB) decreases through cyclosporine treatment and this is mediated by cyclosporine-induced reduction of CREB steady-state levels. These results demonstrate that cyclosporine can inhibit proliferation of acinar cells by targeting the cyclin D1 promoter at the proximal CRE via a reduction of CREB protein abundance.

Animals↗
PubMed2002

Crystal structure of decameric fructose-6-phosphate aldolase from Escherichia coli reveals inter-subunit helix swapping as a structural basis for assembly differences in the transaldolase family.

Fructose-6-phosphate aldolase from Escherichia coli is a member of a small enzyme subfamily (MipB/TalC family) that belongs to the class I aldolases. The three-dimensional structure of this enzyme has been determined at 1.93 A resolution by single isomorphous replacement and tenfold non-crystallographic symmetry averaging and refined to an R-factor of 19.9% (R(free) 21.3%). The subunit folds into an alpha/beta barrel, with the catalytic lysine residue on barrel strand beta 4. It is very similar in overall structure to that of bacterial and mammalian transaldolases, although more compact due to extensive deletions of additional secondary structural elements. The enzyme forms a decamer of identical subunits with point group symmetry 52. Five subunits are arranged as a pentamer, and two ring-like pentamers pack like a doughnut to form the decamer. A major interaction within the pentamer is through the C-terminal helix from one monomer, which runs across the active site of the neighbouring subunit. In classical transaldolases, this helix folds back and covers the active site of the same subunit and is involved in dimer formation. The inter-subunit helix swapping appears to be a major determinant for the formation of pentamers rather than dimers while at the same time preserving importing interactions of this helix with the active site of the enzyme. The active site lysine residue is covalently modified, by forming a carbinolamine with glyceraldehyde from the crystallisation mixture. The catalytic machinery is very similar to that of transaldolase, which together with the overall structural similarity suggests that enzymes of the MipB/TALC subfamily are evolutionary related to the transaldolase family.

Aldehyde-Lyases↗
PubMed2002

Phosphatase and oxygen radical-generating activities of mammalian purple acid phosphatase are functionally independent.

Bone-resorbing osteoclasts and activated macrophages express large amounts of tartrate-resistant acid phosphatase (TRAP), an iron-containing enzyme with unknown biological function. We studied acid phosphatase (AcP) and reactive oxygen species (ROS)-generating activities of recombinant rat TRAP. pH optimum was 4.5 for AcP activity and 6.5 for ROS-generating activity. Replacement of His113 and His216 by site-directed mutagenesis severely inhibited AcP activity, but had no significant effects on ROS-generating activity. Substrate specificity was not affected by the mutations. These results suggest that AcP and ROS-generating activities of TRAP are functionally independent.

Acid Phosphatase↗
PubMed2002

Expression, purification, refolding and crystallization of the carbohydrate-recognition domain of p58/ERGIC-53, an animal C-type lectin involved in export of glycoproteins from the endoplasmic reticulum.

p58/ERGIC-53 is a mammalian calcium-dependent lectin that serves as a glycoprotein-sorting receptor between the endoplasmic reticulum (ER) and the Golgi complex. It is a type I transmembrane protein with two lumenal domains, one of which is a carbohydrate-recognition domain (CRD) and homologous to leguminous lectins. The CRD of p58, the rat homologue of human ERGIC-53, was overexpressed in insect cells and Escherichia coli, purified and crystallized using Li(2)SO(4) as a precipitant. The crystals belong to space group I222, with unit-cell parameters a = 49.6, b = 86.1, c = 128.1 A, and contain one molecule per asymmetric unit, corresponding to a packing density of 2.4 A(3)Da(-1). Knowledge of the structure of p58/ERGIC-53 will provide a starting model for understanding receptor-mediated glycoprotein sorting between the ER and the Golgi.

Animals↗
PubMed2002

Crystal structure of the carbohydrate recognition domain of p58/ERGIC-53, a protein involved in glycoprotein export from the endoplasmic reticulum.

p58/ERGIC-53 is an animal calcium-dependent lectin that cycles between the endoplasmic reticulum (ER) and the Golgi complex and appears to act as a cargo receptor for a subset of soluble glycoproteins exported from the ER. We have determined the crystal structure of the carbohydrate recognition domain (CRD) of p58, the rat homologue of human ERGIC-53, to 1.46 A resolution. The fold and ligand binding site are most similar to those of leguminous lectins. The structure also resembles that of the CRD of the ER folding chaperone calnexin and the neurexins, a family of non-lectin proteins expressed on neurons. The CRD comprises one concave and one convex beta-sheet packed into a beta-sandwich. The ligand binding site resides in a negatively charged cleft formed by conserved residues. A large surface patch of conserved residues with a putative role in protein-protein interactions and oligomerization lies on the opposite side of the ligand binding site. Together with previous functional data, the structure defines a new and expanding class of calcium-dependent animal lectins and provides a starting point for the understanding of glycoprotein sorting between the ER and the Golgi.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil. Implications for mechanism of inhibition and electron transfer.

Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.

Animals↗