PubMed Health⌕ Search

Biomedical subjects

E F Pai

Publications and source records attributed to E F Pai.

At least 19 recordsLinked to original sources
PubMed2000

Insights into ligand binding and catalysis of a central step in NAD+ synthesis: structures of Methanobacterium thermoautotrophicum NMN adenylyltransferase complexes.

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN(+) or NaMN(+) with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD(+), via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD(+) molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved (16)HXGH(19) active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN(+)/SO4(2-) complex of mutant H19A NMNATase.

Amino Acid Sequence↗
PubMed2000

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

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 (M(r), 290,000) bovine milk XDH at 2.1-A resolution and XO at 2.5-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 423Lys 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.

Animals↗
PubMed2000

Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP.

Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3, 5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer beta-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.

Amino Acid Sequence↗
PubMed2000

The active site architecture of Pisum sativum beta-carbonic anhydrase is a mirror image of that of alpha-carbonic anhydrases.

We have determined the structure of the beta-carbonic anhydrase from the dicotyledonous plant Pisum sativum at 1.93 A 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 beta-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 alpha-carbonic anhydrase active site, with the corresponding residues being closely superimposable by a mirror plane. Therefore, despite differing folds, alpha- and beta-carbonic anhydrase have converged upon a very similar active site design and are likely to share a common mechanism.

Amino Acid Sequence↗
PubMed2000

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

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 A, 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.

Amino Acid Sequence↗
PubMed2000

Structural basis for specificity switching of the Src SH2 domain.

The Src SH2 domain binds pYEEI-containing phosphopeptides in an extended conformation with a hydrophobic pocket, which includes ThrEF1, binding Ile(pY +3). Mutating ThrEF1 to tryptophan switches specificity to an Asn(pY +2) requirement, yielding a biological mimic of the Grb2 SH2 domain. Here we show that the Src ThrEF1Trp SH2 domain mutant binds pYVNV phosphopeptides in a beta turn conformation, which, despite differing conformations of the interacting tryptophan, closely resembles the native Grb2/pYVNV cognate peptide binding mode. The ThrEF1Trp substitution therefore switches specificity by physically occluding the pTyr +3 binding pocket and by providing additional interaction surface area for Asn(pY +2). This demonstrates structurally how novel SH2 domain specificities may rapidly evolve through single amino acid substitutions and suggests how new signaling pathways may develop.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 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.

Cloning, Molecular↗
PubMed2000

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

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 P4(1)2(1)2 (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 P2(1) (unit-cell parameters a = 73.0, b = 98.6, c = 73.3 A, gamma = 104.0 degrees ), with four molecules in the asymmetric unit; the crystals diffract to 1.5 A resolution.

Base Sequence↗
PubMed2000

beta-carbonic anhydrase from Pisum sativum: crystallization and preliminary X-ray analysis.

Recombinant beta-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, alpha = beta = gamma = 90 degrees. 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.

Carbonic Anhydrases↗
PubMed2000

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

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 C222(1), 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, beta = 90.9 degrees. 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.

Animals↗
PubMed2000

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

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.

Amino Acid Sequence↗
PubMed1998

Differences in binding modes of enantiomers of 1-acetamido boronic acid based protease inhibitors: crystal structures of gamma-chymotrypsin and subtilisin Carlsberg complexes.

In order to probe the structural basis of stereoselectivity in the serine protease family, a series of enantiomeric boronic acids RCH2CH(NHCOCH3)B(OH)2 has been synthesized and kinetically characterized as transition-state analog inhibitors using alpha-chymotrypsin and subtilisin Carlsberg as model systems. When the R-substituent in this series was changed from a p-chlorophenyl to a 1-naphthyl group, alpha-chymotrypsin, but not subtilisin, reversed its usual preference for l-enantiomers and bound more tightly to the D-enantiomer [Martichonok, V., & Jones, J. B. (1996) J. Am. Chem. Soc. 118, 950-958]. The structural factors responsible for the differences in stereoselectivity between the two enzymes have been explored by X-ray crystallographic examination of subtilisin Carlsberg and gamma-chymotrypsin complexes of the L- and D-enantiomers of p-chlorophenyl and 1-naphthyl boronic acid derivatives. In both enzymes, the L-isomers of the inhibitors, which are more closely related to the natural L-amino acid substrates, form tetrahedral adducts, covalently linking the central boron atom and Ogamma of the catalytic serine. The d-isomers, however, differ in the way they interact with subtilisin or gamma-chymotrypsin. With subtilisin, both the D-p-chlorophenyl and D-1-naphthyl inhibitor complexes form covalent Ser Ogamma-to-boron bonds, but with gamma-chymotrypsin, the same inhibitors lead to novel tetrahedral adducts covalently linking both Ser195 Ogamma and His57 Nepsilon2 covalently via the boron atom.

Binding Sites↗
PubMed1998

A thioredoxin fusion protein of VanH, a D-lactate dehydrogenase from Enterococcus faecium: cloning, expression, purification, kinetic analysis, and crystallization.

The gene encoding the vancomycin resistance protein VanH from Enterococcus faecium, a D-lactate dehydrogenase, has been cloned into a thioredoxin expression system (pTRxFus) and expressed as a fusion protein. The use of several other expression systems yielded only inclusion bodies from which no functional protein could be recovered. Experiments to remove the thioredoxin moiety by enterokinase cleavage at the engineered recognition site under a variety of conditions resulted in nonspecific proteolysis and inactivation of the protein. The intact fusion protein was, therefore, used for kinetic studies and crystallization trials. It has been purified to greater than 90% homogeneity by ammonium sulfate precipitation followed by phenyl Sepharose chromatography. Based on k(cat)/KM for pyruvate, it is 20% as active as native VanH. Michaelis constants for NADPH, NADH, and pyruvate, of approximately 3.5 microM, 19.0 microM, and 1.5 mM, respectively, were comparable to those reported for the native VanH (Bugg TDH et al., 1991, Biochemistry 30:10408-10415). Like native VanH, maximum activity of the fusion protein requires the presence of an anion (phosphate or acetate), however, in addition, a strongly reducing environment is needed for optimal efficacy. Competitive inhibition constants for ADP-ribose, NAD+, and oxamate have also been determined. Crystallization by hanging drop vapor diffusion produced two different crystal forms, one hexagonal and the other tetragonal. Flash-frozen crystals of the tetragonal form diffracted to 3.0 A resolution at a synchrotron radiation source.

Amino Acid Sequence↗
PubMed1998

The alpha and beta of turning on a molecular switch.

The crystal structures of RCC1 and the Sec7 domain of human Arno, nucleotide exchange factors for the Ras-related GTPases Ran and ARF, reveal two very different folds, the former a seven-bladed beta-propeller, the latter a capped right-handed superhelix. Both are also unrelated to the folds of Mss4 and elongation factor Ts, nucleotide exchange factors for Rab and elongation factor Tu.

Amino Acid Sequence↗
PubMed1997

The structure of enzyme IIAlactose from Lactococcus lactis reveals a new fold and points to possible interactions of a multicomponent system.

BACKGROUND: The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) is responsible for the binding, transmembrane transport and phosphorylation of numerous sugar substrates. The system is also involved in the regulation of a variety of metabolic and transcriptional processes. The PTS consists of two non-specific energy coupling components, enzyme I and a heat stable phosphocarrier protein (HPr), as well as several sugar-specific multiprotein permeases known as enzymes II. In most cases, enzymes IIA and IIB are located in the cytoplasm, while enzyme IIC acts as a membrane channel. Enzyme IIAlactose belongs to the lactose/cellobiose-specific family of enzymes II, one of four functionally and structurally distinct groups. The protein, which normally functions as a trimer, is believed to separate into its subunits after phosphorylation. RESULTS: The crystal structure of the trimeric enzyme IIAlactose from Lactococcus lactis has been determined at 2.3 A resolution. The subunits of the enzyme, related to each other by the inherent threefold rotational symmetry, possess interesting structural features such as coiled-coil-like packing and a methionine cluster. The subunits each comprise three helices (I, II and III) and pack against each other forming a nine-helix bundle. This helical bundle is stabilized by a centrally located metal ion and also encloses a hydrophobic cavity. The three phosphorylation sites (His78 on each monomer) are located in helices III and their sidechains protrude into a large groove between helices I and II of the neighbouring subunits. A model of the complex between phosphorylated HPr and enzyme IIAlactose has been constructed. CONCLUSIONS: Enzyme IIAlactose is the first representative of the family of lactose/cellobiose-specific enzymes IIA for which a three-dimensional structure has been determined. Some of its structural features, like the presence of two histidine residues at the active site, seem to be common to all enzymes no overall structural homology is observed to any PTS proteins or to any other proteins in the Protein Data Bank. Enzyme IIAlactose shows surface complementarity to the phosphorylated form of HPr and several energetically favourable interactions between the two molecules can be predicted.

Amino Acid Sequence↗
PubMed1997

Glutathione reductase turned into trypanothione reductase: structural analysis of an engineered change in substrate specificity.

Trypanosoma and Leishmania, pathogens responsible for diseases such as African sleeping sickness, Chagas' heart disease, or Oriental sore, are two of the very few genera that do not use the ubiquitous glutathione/glutathione reductase system to keep a stable cellular redox balance. Instead, they rely on trypanothione and trypanothione reductase to protect them from oxidative stress. Trypanothione reductase (TR) and the corresponding host enzyme, human red blood cell glutathione reductase (GR), belong to the same flavoprotein family. Despite their closely related three-dimensional structures and although their natural substrates share the common structural glutathione core, the two enzymes are mutually exclusive with respect to their disulfide substrates. This makes the parasite enzyme a potential target for antitrypanosomal drug design. While a large body of structural data on GR complexes is available, information on TR-ligand interactions is very limited. When the two amino acid changes Ala34Glu and Arg37Trp are introduced into human GR, the resulting mutant enzyme (GRTR) prefers trypanothione 700-fold over its original substrate, effectively converting a GR into a TR [Bradley, M., Bücheler, U. S., & Walsh, C. T. (1991) Biochemistry 30, 6124-6127]. The crystal structure of GRTR has been determined at 2.3 A resolution and refined to a crystallographic R factor of 20.9%. We have taken advantage of the ease with which ligand complexes can be produced in GR crystals, a property that extends to the isomorphous GRTR crystals, and have produced and analyzed crystals of GRTR complexes with glutathione, trypanothione, glutathionylspermidine and of a true catalytic intermediate, the mixed disulfide between trypanothione and the enzyme. The corresponding molecular structures have been characterized at resolutions between 2.3 and 2.8 A with R factors ranging from 17.1 to 19.7%. The results indicate that the Ala34Glu mutation causes steric hindrance leading to a large displacement of the side chain of Arg347. This movement combined with the change in charge introduced by the mutations modifies the binding cavity, forcing glutathione to adopt a nonproductive binding mode and permitting trypanothione and to a certain degree also the weak substrate glutathionylspermidine to assume a productive mode.

Animals↗
PubMed1996

The Grb2-mSos1 complex binds phosphopeptides with higher affinity than Grb2.

Epidermal growth factor (EGF) stimulation leads to autophosphorylation of the epidermal growth factor receptor (EGFR) and tyrosine phosphorylation of Shc. The Grb2 SH2 domain binds to Tyr1068 of EGFR and Tyr317 of Shc while its SH3 domains bind to mSos1. Therefore, EGF treatment potentially results in the formation of several multimeric signaling complexes, including EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc-Grb2-mSos1, linking the receptor to activation of the Ras GTPase. We have purified Grb2, mSos1, and the Grb2-mSos1 complex to high homogeneity, and used these isolated proteins to obtain binding affinities of mSos1 for Grb2 and of either Grb2 or Grb2-mSos1 for phosphotyrosine-containing peptides. mSos1 bound Grb2 with a KD of 0.4 microM; the stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopeptide bound Grb2 with a KD of 0.7 microM, whereas the Shc-derived phosphopeptide bound Grb2 with a KD of 0.2 microM. Since Grb2 exists in a stable complex with mSos1, and both proteins can exist in a constitutive complex in unstimulated cells, we performed phosphopeptide binding studies on the Grb2-mSos1 complex to gain a better understanding of binding events in the intact cell. Grb2-mSos1 bound to both EGFR- and Shc-derived phosphopeptides with higher affinities (KD of 0.3 microM and 31 nM, respectively) than Grb2 alone. These findings suggest that the proximity of mSos1 to Grb2 in the complex can influence the interactions of the Grb2 SH2 domain with phosphopeptides and raise the possibility that in the Grb2-mSos1 complex the SH2 and SH3 domains of Grb2 are not independent of each other but may be indirectly linked by mSos1.

Adaptor Proteins, Signal Transducing↗