Doreen Dobritzsch

High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis : Implications for substrate activation in pyruvate decarboxylases

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 Å resolution and refined to a crystallographicR-factor of 16.2% and R free of 19.7%. The subunit consists of three domains, all of the α/β type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil

Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH‐dependent reduction of uracil and thymine to the corresponding 5,6‐dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5‐fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2× 111 kDa) determined at 1.9 Å resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe–4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe–S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5‐fluorouracil reveals the architecture of the substrate‐binding sites and residues responsible for recognition and binding of the drug.

A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast.

In humans, β‐alanine (BAL) and the neurotransmitter γ‐aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiaeUGA1 and Schizosaccharomyces pombeUGA1 were subcloned, over‐expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5′‐phosphate is needed for enzymatic activity and α‐ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (Vmax/Km = 0.78 U·mg−1·mm−1), but can also use GABA (Vmax/Km = 0.42 U·mg−1·mm−1), while SkUga1p only uses GABA (Vmax/Km = 4.01 U·mg−1·mm−1). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently ‘distinguish’ between the two reactions in a single cell. It is likely that upon duplication ∼200 million years ago, a specialized Uga1p evolved into a ‘novel’ transaminase enzyme with broader substrate specificity.

The Crystal Structures of Dihydropyrimidinases Reaffirm the Close Relationship between Cyclic Amidohydrolases and Explain Their Substrate Specificity.

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 Å, respectively. Both enzymes have a (β/α)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller β-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the β-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-β-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.

Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases.

β-Alanine synthase (βAS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, βASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of βAS from Saccharomyces kluyveri to a resolution of 2.7 Å. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of βAS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed α/β-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by βAS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.

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 Å 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 α/β-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.

Novel tetramer assembly of pyruvate decarboxylase from brewer's yeast observed in a new crystal form

© 1997 Federation of European Biochemical Societies.

Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients.

Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5-8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated.

The Crystal Structure of Beta-Alanine Synthase from Drosophila Melanogaster Reveals a Homooctameric Helical Turn-Like Assembly.

Beta-alanine synthase (betaAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate to the corresponding beta-amino acids. betaASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote betaAS subfamily, we determined the crystal structure of Drosophila melanogaster betaAS to 2.8 A resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central beta-sandwich with a layer of alpha-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster betaAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.

Crystal structure of an arthritogenic anticollagen immune complex

OBJECTIVE In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation. METHODS Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide. RESULTS The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope. CONCLUSION The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures.