Showing papers by "Emil F. Pai published in 2000"

Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion

Abstract: Mammalian xanthine oxidoreductases, which catalyze the last two steps in the formation of urate, are synthesized as the dehydrogenase form xanthine dehydrogenase (XDH) but can be readily converted to the oxidase form xanthine oxidase (XO) by oxidation of sulfhydryl residues or by proteolysis Here, we present the crystal structure of the dimeric (Mr, 290,000) bovine milk XDH at 21-A resolution and XO at 25-A resolution and describe the major changes that occur on the proteolytic transformation of XDH to the XO form Each molecule is composed of an N-terminal 20-kDa domain containing two iron sulfur centers, a central 40-kDa flavin adenine dinucleotide domain, and a C-terminal 85-kDa molybdopterin-binding domain with the four redox centers aligned in an almost linear fashion Cleavage of surface-exposed loops of XDH causes major structural rearrangement of another loop close to the flavin ring (Gln 423—Lys 433) This movement partially blocks access of the NAD substrate to the flavin adenine dinucleotide cofactor and changes the electrostatic environment of the active site, reflecting the switch of substrate specificity observed for the two forms of this enzyme

Structural proteomics of an archaeon.

Abstract: A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

The active site architecture of Pisum sativum β-carbonic anhydrase is a mirror image of that of α-carbonic anhydrases

Abstract: We have determined the structure of the β–carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 Å resolution, using a combination of multiple anomalous scattering off the active site zinc ion and non-crystallographic symmetry averaging. The mol– ecule assembles as an octamer with a novel dimer of dimers of dimers arrangement. Two distinct patterns of conservation of active site residues are observed, implying two potentially mechanistically distinct classes of β–carbonic anhydrases. The active site is located at the interface between two monomers, with Cys160, His220 and Cys223 binding the catalytic zinc ion and residues Asp162 (oriented by Arg164), Gly224, Gln151, Val184, Phe179 and Tyr205 interacting with the substrate analogue, acetic acid. The substrate binding groups have a one to one correspondence with the functional groups in the α–carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, α- and β–carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.

Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase.

Abstract: Orotidine 5′-monophosphate decarboxylase catalyzes the conversion of orotidine 5′-monophosphate to uridine 5′-monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5′-monophosphate-complexed forms have been determined at 1.8 and 1.5 Å, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5′-monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.

Penicillopepsin-JT2, a recombinant enzyme from Penicillium janthinellum and the contribution of a hydrogen bond in subsite S3 to k(cat).

Abstract: The nucleotide sequence of the gene (pepA) of a zymogen of an aspartic proteinase from Penicillium janthinellum with a 71% identity in the deduced amino acid sequence to penicillopepsin (which we propose to call penicillopepsin-JT1) has been determined. The gene consists of 60 codons for a putative leader sequence of 20 amino acid residues, a sequence of about 150 nucleotides that probably codes for an activation peptide and a sequence with two introns that codes for the active aspartic proteinase. This gene, inserted into the expression vector pGPT-pyrG1, was expressed in an aspartic proteinase-free strain of Aspergillus niger var. awamori in high yield as a glycosylated form of the active enzyme that we call penicillopepsin-JT2. After removal of the carbohydrate component with endoglycosidase H, its relative molecular mass is between 33,700 and 34,000. Its kinetic properties, especially the rate-enhancing effects of the presence of alanine residues in positions P3 and P2' of substrates, are similar to those of penicillopepsin-JT1, endothiapepsin, rhizopuspepsin, and pig pepsin. Earlier findings suggested that this rate-enhancing effect was due to a hydrogen bond between the -NH- of P3 and the hydrogen bond accepting oxygen of the side chain of the fourth amino acid residue C-terminal to Asp215. Thr219 of penicillopepsin-JT2 was mutated to Ser, Val, Gly, and Ala. Thr219Ser showed an increase in k(cat) when a P3 residue was present in the substrate, which was similar to that of the wild-type, whereas the mutants Thr219Val, Thr219Gly, and Thr219Ala showed no significant increase when a P3 residue was added. The results show that the putative hydrogen bond alone is responsible for the increase. We propose that by locking the -NH- of P3 to the enzyme, the scissile peptide bond between P1 and P1' becomes distorted toward a tetrahedral conformation and becomes more susceptible to nucleophilic attack by the catalytic apparatus without the need of a conformational change in the enzyme.

Purification, crystallization and preliminary X-ray diffraction studies of xanthine dehydrogenase and xanthine oxidase isolated from bovine milk.

Abstract: Xanthine dehydrogenase catalyzes the oxidation of hypoxanthine to xanthine and the further oxidation of xanthine to uric acid. The enzyme is the target of the anti-gout drug allopurinol and its involvement in postischemic reperfusion injury is presently being defined. Each subunit of the homodimeric 290 kDa enzyme contains four cofactors: one Mo-pterin, two [2Fe–2S] clusters and one FAD. Both the dehydrogenase (XDH) and the proteolytically modified oxidase form (XO) of the enzyme from bovine milk have been crystallized. XO crystals belong to space group C2221, with unit-cell parameters a = 116.3, b = 164.4, c = 153.2 A at room temperature and a = 117.8, b = 165.4, c = 154.5 A when flash-frozen. They allow data collection to 3.3 and 2.5 A, respectively. In addition, a data set was collected from frozen XDH crystals and processed to 2.1 A. These crystals belong to space group C2, with unit-cell parameters a = 169.9, b = 124.8, c = 148.6 A, β = 90.9°. The unit-cell volumes and Matthews parameters are similar for the two crystal forms. There is one monomer per asymmetric unit in the XO crystals and a complete native dimer per asymmetric unit in the XDH crystals.

Fab'-epitope complex from the hiv-1 cross-neutralizing monoclonal antibody 2f5

Abstract: The crystal structure of the Fab' fragment of Mab 2F5, a potent neutralizer of both laboratory strains and primary clinical isolates of most clades of HIV-1, both uncomplexed and complexed with the largely conserved peptide sequence ELDKWAS of the viral envelope protein gp41, has been elucidated and the characteristics of peptide-protein interactions determined. Having regard to such determination, the peptide-mimetics are constrained in the three-dimensional structure to provide an increased immunogenicity to the epitope sequence.

Purification, crystallization and preliminary X-ray study of orotidine 5′-monophosphate decarboxylase

Abstract: Orotidine-5′-monophosphate decarboxylase (ODCase) from Methanobacterium thermoautotrophicum has been crystallized with and without the inhibitor 6-azaUMP by the vapour-diffusion method. In the absence of the inhibitor, the protein crystallizes in space group P41212 (unit-cell parameters a = b = 56.9, c = 124.5 A) with one molecule per asymmetric unit; the crystals diffract to 1.8 A resolution. In the presence of the inhibitor, the protein crystals are monoclinic, space group P21 (unit-cell parameters a = 73.0, b = 98.6, c = 73.3 A, γ = 104.0°), with four molecules in the asymmetric unit; the crystals diffract to 1.5 A resolution.

β-Carbonic anhydrase from Pisum sativum: crystallization and preliminary X-ray analysis

Abstract: Recombinant β-carbonic anhydrase from the garden pea, Pisum sativum, was purified to homogeneity and crystallized. Crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 136.3, b = 142.5, c = 201.4 A, α = β = γ = 90°. Crystals typically diffracted anisotropically, with a maximal resolution of 2.0 A in the strongest direction. The calculated Matthews parameter predicts approximately eight molecules in the asymmetric unit, consistent with previous reports of the molecule being an octamer. However, examination of the self-rotation function revealed no fourfold symmetry axis and multiple weak twofold axes perpendicular to the crystallographic c axis, indicating that the oligomerization arrangement is not that of a 422 octamer.