Biomedical subjects

D L Ollis

Publications and source records attributed to D L Ollis.

At least 37 records · Page 2Linked to original sources

PubMed1998

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains.

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.

Amino Acid Sequence↗

PubMed1997

The two opposing activities of adenylyl transferase reside in distinct homologous domains, with intramolecular signal transduction.

Adenylyl transferase (ATase) is the bifunctional effector enzyme in the nitrogen assimilation cascade that controls the activity of glutamine synthetase (GS) in Escherichia coli. This study addresses the question of whether the two antagonistic activities of ATase (adenylylation and deadenylylation) occur at the same or at different active sites. The 945 amino acid residue ATase has been truncated in two ways, so as to produce two homologous polypeptides corresponding to amino acids 1-423 (AT-N) and 425-945 (AT-C). We demonstrate that ATase has two active sites; AT-N carries a deadenylylation activity and AT-C carries an adenylylation activity. Glutamine activates the adenylylation reaction of the AT-C domain, whereas alpha-ketoglutarate activates the deadenylylation reaction catalysed by the AT-N domain. With respect to the regulation by the nitrogen status monitor PII, however, the adenylylation domain appears to be dependent on the deadenylylation domain: the deadenylylation activity of AT-N depends on PII-UMP and is inhibited by PII. The adenylylation activity of AT-C is independent of PII (or PII-UMP), whereas in the intact enzyme PII is required for this activity. The implications of this intramolecular signal transduction for the prevention of futile cycling are discussed.

Amino Acid Sequence↗

PubMed1997

Crystal structure of cytoplasmic Escherichia coli peptidyl-prolyl isomerase: evidence for decreased mobility of loops upon complexation.

The structure of the unliganded form of the Escherichia coli cytoplasmic peptidyl-prolyl isomerase (ppiB gene product) in a new crystal form was determined by the molecular replacement method and refined to an R-factor of 16.1% at 2.1 A resolution. The enzyme crystallized in the orthorhombic C2221 space group with unit cell dimensions of a=44.7 A, b=68.2 A and c=102.0 A. Comparison with the reported structure of the enzyme complexed with the tripeptide substrate succinyl-Ala-Pro-Ala-p-nitroanilide revealed subtle changes that occur upon complex formation. There is evidence to suggest that two surface loops have significantly reduced mobility in the complexed structure.

Amino Acid Isomerases↗

PubMed1997

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly.

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Alleles↗

PubMed1997

Structure of ferric soybean leghemoglobin a nicotinate at 2.3 A resolution.

Soybean leghemoglobin a is a small (16 kDa) protein facilitating the transport of O(2) to respiring N(2)-fixing bacteria at low free-O(2) tension. The crystal structure of soybean ferric leghemoglobin a nicotinate has been refined at 2.3 A resolution. The final R factor is 15.8% for 6877 reflections between 6.0 and 2.3 A. The structure of soybean leghemoglobin a (143 residues) is closely similar to that of lupin leghemoglobin II (153 residues), the proteins having 82 identical residues when the sequences are aligned. The new structure provides support for the conclusion that the unique properties of leghemoglobin arise principally from a heme pocket considerably larger and more flexible than that of myoglobin, a strongly ruffled heme group, and a proximal histidine orientation more favourable to ligand binding.

Journal Article↗

PubMed1996

The role of the T-loop of the signal transducing protein PII from Escherichia coli.

The 3D structure of PII, the central protein that controls the level of transcription and the enzymatic activity of glutamine synthetase in enteric bacteria revealed that residues 37-55 form the "T' loop, part of which protrudes from the core of the protein. Within this loop are the only two tyrosine residues that occur in the polypeptide, and one of them, Tyr-51, has been shown by chemical modification studies to be the site of uridylylation. Since tyrosine at position 46 is conserved in all known PII proteins, oligonucleotide directed mutagenesis was used to investigate the role of the two residues. Changing Tyr-51 to phenylalanine or serine abolished uridylylation. Altering tyrosine at position 46 to phenylalanine affected the rate of uridylylation of the protein. This latter mutation does not alter the structure of PII but the reduction in the uridylylation efficiency suggests a role for this residue in recognition and binding of the sensor enzyme uridylyl transferase.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary X-ray diffraction studies of new crystal forms of Escherichia coli P(II) complexed with various ligands.

New crystals of the signal-transducing protein P(II) have been obtained in the presence of a number of different effector ligands. Various crystal forms are observed depending on the nature of the ligand(s). Co-crystallization with 2-ketoglutarate, glutamate and pyrophosphate produces hexagonal crystals similar to the wild type, ATP yields cubic crystals and ATP in conjunction with 2-ketoglutarate or glutamate yields orthorhombic crystal forms. All of the above crystals have been characterized by X-ray diffraction analysis. The hexagonal crystals belong to space group P6(3), cubic crystals to either I23 or I2(1)3 and orthorhombic crystals to I222. A molecular-replacement solution for the P(II)/ATP/2-ketoglutarate crystals has been obtained giving us an initial model for a trimer in the orthorhombic crystal form.

Journal Article↗

PubMed1996

Structural and sequence comparisons of quinone oxidoreductase, zeta-crystallin, and glucose and alcohol dehydrogenases.

Quinone oxidoreductase, zeta-crystallin, glucose dehydrogenase, and alcohol dehydrogenase belong to a superfamily of medium-chain dehydrogenase/reductases. The crystal structures of Escherichia coli quinone oxidoreductase (QOR) and Thermoplasma acidophilum glucose dehydrogenase have recently been determined and are compared here with the well-known structure of horse liver alcohol dehydrogenase. A structurally based comparison of these three enzymes confirms that they possess extensive overall structural homology despite low sequence identity. The most significant difference is the absence of the catalytic and structural zinc ions in QOR. A multiple structure-based sequence alignment has been constructed for the three enzymes and extended to include zeta-crystallin, an eye lens structural protein with quinone oxidoreductase activity and high sequence identity to E. coli quinone oxidoreductase. Residues which are important for catalysis have been altered and the functions and activities of the enzymes have diverged, illustrating a classic example of divergent evolution among a superfamily of enzymes.

Alcohol Dehydrogenase↗

PubMed1996

X-ray structure of the signal transduction protein from Escherichia coli at 1.9 A.

The structure of the bacterial signal transduction protein P(II) has been refined to an R factor of 13.2% using 3sigma data between 10 and 1.9 A. The crystals exhibited twinning by merohedry and X-ray intensities were corrected using the method of Fisher & Sweet [Fisher & Sweet (1980). Acta Cryst. A36, 755-760] prior to refinement. Our earlier 2.7 A structure [Cheah, Carr, Suffolk, Vasudevan, Dixon & Ollis (1994). Structure, 2, 981-990] served as a starting model. P(II) is a trimeric molecule, each subunit has a mass of 12.4 kDa and contains 112 amino-acid residues. The refined model includes all 1065 protein atoms per subunit plus 312 water molecules. The high-resolution refinement confirms the correctness of our 2.7 A model, although it leads to a redefinition of the extent of various secondary-structural elements. The monomeric structure of P(II) exhibits an interlocking double betaalphabeta fold. This is a stable fold found in a number of proteins with diverse functions. The association of the protein into a trimer leads to a new structure which we describe in detail. The effects of crystal packing forces are discussed and potential interaction sites with other proteins and effector molecules are identified.

Journal Article↗

PubMed1995

Crystal structure of Escherichia coli QOR quinone oxidoreductase complexed with NADPH.

The crystal structure of the homodimer of quinone oxidoreductase from Escherichia coli has been determined using the multiple isomorphous replacement method at 2.2 A resolution and refined to an R-factor of 14.1% The crystallographic asymmetric unit contains one functional dimer with the two subunits being related by a non-crystallographic 2-fold symmetry axis. The model consists of two polypeptide chains (residues 2 through 327), one NADPH molecule and one sulphate anion per subunit, and 432 water molecules. Each subunit consists of two domains: a catalytic domain and a nucleotide-binding domain with the NADPH co-factor bound in the cleft between domains. Quinone oxidoreductase has an unusual nucleotide-binding fingerprint motif consisting of the sequence AXXGXXG. The overall structure of quinone oxidoreductase shows strong structural homology to that of horse liver alcohol dehydrogenase.

Amino Acid Sequence↗

PubMed1995

A theoretical study of substrate-induced activation of dienelactone hydrolase.

Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate pathway, catalyses the hydrolysis of dienelactone to maleylacetate. DLH is unusual because it is the only known naturally occurring enzyme which contains the catalytic triad Cys ... His ... Asp. This triad has previously been created artificially in the mutant serine proteases, thiol subtilisin and thiol trypsin. In both cases the mutant enzymes exhibited activities several orders of magnitude lower than the wild type enzymes; the low reactivity has generally been attributed to the inability of these enzymes to form a catalytically active thiolate anion (Cys- ... His+ ... Asp-). The crystal structure of DLH suggests that the native enzyme exists predominantly in a catalytically inert configuration; the triad cysteine is neutral and points away from the active site binding cleft. However, a crystallographic analysis of C123S DLH complexed with an isostructural inhibitor (dienelactam) indicates that substrate binding induces a prototropic rearrangement of the active site prior to catalysis which results in the formation of a highly nucleophilic thiolate anion. We have performed ab initio SCF/MP2 calculations on a relatively small portion of the active site of DLH to examine the details of this activation process. Our calculations provide supporting evidence that the conformational changes observed in the crystal structure due to inhibitor (or substrate) binding facilitate the formation of a reactive thiolate anion. In particular, substrate binding alters the position of Glu36; the carboxylate side chain of Glu36 is pushed towards C123 enabling it to abstract the thiol proton thus creating a catalytically active thiolate anion.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Structure of the Escherichia coli signal transducing protein PII.

BACKGROUND: In Gram-negative proteobacteria, the nitrogen level in the cell is reflected by the uridylylation status of a key signal transducing protein, PII. PII modulates the activity of glutamine synthetase (GS) through its interaction with adenylyl transferase and it represses the expression of GS by acting in concert with nitrogen regulatory protein II. RESULTS: The three-dimensional structure of the Escherichia coli PII trimer has been determined at 2.7 A resolution. PII shows a low level of structural similarity to a broad family of alpha/beta proteins and contains a double beta alpha beta motif. The PII trimer contains three beta-sheets, each of which is composed of strands from each of the three monomers. These are surrounded by six alpha-helices. CONCLUSIONS: The structure of PII suggests potential regions of interaction with other proteins and serves as an initial step in understanding its signal transducing role in nitrogen regulation.

Amino Acid Sequence↗

PubMed1994

Crystallization and preliminary X-ray diffraction studies on a soluble Escherichia coli quinone oxidoreductase.

Single crystals suitable for X-ray diffraction studies have been obtained from a soluble Escherichia coli NAD(P)H-dependent quinone oxidoreductase (QOR) of molecular mass 35 kDa. Crystals were grown by the vapour diffusion method in the presence of NADPH and diffract strongly to beyond 2.0 A. The space group was determined to be P2(1)2(1)2(1) with unit cell dimensions of a = 107.44 A, b = 104.06 A, c = 77.45 A. There is one dimer in the asymmetric unit.

Crystallization↗

PubMed1994

Crystallization and low temperature diffraction studies of the DNA binding domain of the single-stranded DNA binding protein from Escherichia coli.

The DNA binding domain of the single-stranded DNA binding protein from Escherichia coli has been overproduced, purified and crystallized in a form suitable for X-ray diffraction studies. Crystals were produced by dialysis against low ionic strength buffer at high pH. The crystals belong to space group I222 or I2(1)2(1)2(1) with a = 82.47 A, b = 65.27 A and c = 46.50 A. Data were collected at several temperatures and a significant improvement in data quality was observed with a decrease in temperature. On occasion, with the decrease in temperature, a transformation to a primitive space group was observed and temperature appears to play a key role in this transformation. A complete native data set has been collected to 2.57 A at -15 degrees C.

Bacterial Proteins↗

PubMed1994

Escherichia coli PII protein: purification, crystallization and oligomeric structure.

The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.

Bacterial Proteins↗

PubMed1992

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.

Acetylcholinesterase↗

PubMed1987

Crystal structure of muconate lactonizing enzyme at 3 A resolution.

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.

Binding Sites↗

PubMed1986

A domain of the Klenow fragment of Escherichia coli DNA polymerase I has polymerase but no exonuclease activity.

The Klenow fragment of DNA polymerase I from Escherichia coli has two enzymatic activities: DNA polymerase and 3'-5' exonuclease. The crystal structure showed that the fragment is folded into two distinct domains. The smaller domain has a binding site for deoxynucleoside monophosphate and a divalent metal ion that is thought to identify the 3'-5' exonuclease active site. The larger C-terminal domain contains a deep cleft that is believed to bind duplex DNA. Several lines of evidence suggested that the large domain also contains the polymerase active site. To test this hypothesis, we have cloned the DNA coding for the large domain into an expression system and purified the protein product. We find that the C-terminal domain has polymerase activity (albeit at a lower specific activity than the native Klenow fragment) but no measurable 3'-5' exonuclease activity. These data are consistent with the hypothesis that each of the three enzymatic activities of DNA polymerase I from E. coli resides on a separate protein structural domain.

Binding Sites↗