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D Ollis

Publications and source records attributed to D Ollis.

13 recordsLinked to original sources
PubMed1993

Catalysis by dienelactone hydrolase: a variation on the protease mechanism.

Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation.

Carboxylic Ester Hydrolases↗
PubMed1993

Substrate-induced activation of dienelactone hydrolase: an enzyme with a naturally occurring Cys-His-Asp triad.

The Cys-His-Asp catalytic triad found in dienelactone hydrolase (DLH) is unusual for several reasons. It has not been observed in other hydrolytic enzymes and it is virtually inactive when it is produced by site-directed mutagenesis in the proteases. We propose a model to explain why this triad is catalytically active in DLH but not in the proteases. In the resting state of DLH, His202 forms an ion pair with Asp171 and Cys123 exists as a thiol. The resting state thiol does not interact with His202 in the active site but instead forms a hydrogen bond with Glu36 in the interior of the molecule. In the absence of substrate, Glu36 is also ion paired with Arg206. When substrate binds, Arg206 forms a second ion pair with the anionic substrate and the Arg206/Glu36 ion pair weakens. The destabilized Glu36 carboxylate shifts towards and deprotonates the Cys123 thiol, thereby activating the nucleophile. As the thiolate anion is not energetically favoured in the hydrophobic interior of the enzyme, it swings into the active site where it can be stabilized by the His202 imidazolium and the dipole of helix C. The Cys123 thiolate which now lies adjacent to the acyl carbon of the substrate, is thus generated only in the presence of substrate. The mode of thiolate activation reduces the susceptibility of DLH towards thiol alkylating agents.

Amino Acid Sequence↗
PubMed1991

Thiol protease-like active site found in the enzyme dienelactone hydrolase: localization using biochemical, genetic, and structural tools.

The active site of dienelactone hydrolase (DLH), a microbial enzyme of the beta-ketoadipate pathway, has been conclusively located using a combination of crystallographic, biochemical, and genetic techniques. DLH hydrolyzes a dienelactone to maleylacetate and has esterase activity on p-nitrophenyl acetate and trans-cinnamoyl imidazole. The identification of Cys-123 as containing the essential thiol confirms the localization of the active site as suggested by the crystal structure of DLH, and disproves an earlier hypothesis regarding its location. Two mutant proteins have been engineered in which Cys-123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH), respectively. C123S DLH (Km = 9900 +/- 2300 microM; Vmax = 4.4 +/- 0.8 mumol/min-mg) displays burst kinetics with p-nitrophenyl acetate and is 10% as active as DLH (Km = 170 +/- 7 microM; Vmax = 21.1 +/- 0.4 mumol/min-mg). C123A DLH is inactive. The structures of DLH, C123S DLH, and C123A DLH have been refined at 1.8, 2.2, and 2.0 A, respectively. Comparison of the structures of these proteins demonstrates that the only differences between them are centered at residue 123. The structures of the active sites of DLH, papain, and subtilisin are similar and are suggestive of the three enzymes having evolved convergently to similar active sites with similar enzymic mechanisms.

Base Sequence↗
PubMed1990

Refined structure of dienelactone hydrolase at 1.8 A.

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8 A resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the carboxyl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014 A and the error in atomic co-ordinates is estimated to be 0.15 A. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the beta-ketoadipate pathway found in bacteria and fungi. DLH is an alpha/beta protein containing seven helices and eight strands of beta-pleated sheet. A single 4-turn 3(10)-helix is seen. The active-site Cys123 residues at the N-terminal end of an alpha-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The beta-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central beta-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the gamma-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.

Amino Acid Sequence↗
PubMed1988

X-ray crystallographic structure of dienelactone hydrolase at 2.8 A.

The structure of dienelactone hydrolase, an enzyme of the beta-ketoadipate pathway, has been determined at 2.8 A resolution using multiple isomorphous replacement techniques. An unambiguous assignment of C alpha atoms to electron density has been accomplished and a preliminary identification of the active site made. Dienelactone hydrolase is an alpha/beta protein consisting of an eight-stranded beta-pleated sheet with seven parallel strands, surrounded by seven helices. Preliminary enzyme inactivation data and an examination of the atomic model have implicated cysteine 123, histidine 202 and aspartate 171 with the active site of the enzyme. It is believed that the enzymic mechanism of dienelactone hydrolase may be similar to that of the thiol and serine proteases.

Amino Acid Sequence↗
PubMed1985

Electrostatic field of the large fragment of Escherichia coli DNA polymerase I.

The electrostatic field of the large fragment of Escherichia coli DNA polymerase I (Klenow fragment) has been calculated by the finite difference procedure on a 2 A grid. The potential field is substantially negative at physiological pH (reflecting the net negative charge at this pH). The largest regions of positive potential are in the deep crevice of the C-terminal domain, which is the proposed binding site for the DNA substrate. Within the crevice, the electrostatic potential has a partly helical form. If the DNA is positioned to fulfil stereochemical requirements, then the positive potential generally follows the major groove and (to a lesser extent) the negative potential is in the minor groove. Such an arrangement could stabilize DNA configurations related by screw symmetry. The histidine residues of the Klenow fragment give the positive field of the groove a sensitivity to relatively small pH changes around neutrality. We suggest that the histidine residues could change their ionization states in response to DNA binding, and that this effect could contribute to the protein-DNA binding energy.

DNA Polymerase I↗
PubMed1985

Low resolution crystal structure of muconolactone isomerase. A decamer with a 5-fold symmetry axis.

Muconolactone isomerase from Pseudomonas putida crystallizes from sodium sulfate solution in space group P2(1) (a = 65.84 A, b = 105.70 A, c = 77.20 A, beta = 90.5 degrees) with ten 11,000 Mr subunits per asymmetric unit. The 7 A resolution crystal structure was solved by single isomorphous replacement followed by 10-fold symmetry averaging. The decameric enzyme has an uncommon non-crystallographic 5-fold symmetry axis and a large cavity in its center.

Bacterial Proteins↗
PubMed1985

Crystal structure of muconate lactonizing enzyme at 6.5 A resolution.

We have obtained crystals of Pseudomonas putida muconate lactonizing enzyme. They diffract to better than 2.4 A resolution and have two monomers in the asymmetric unit, related by a non-crystallographic 2-fold axis. The cell dimensions are 139.3 A X 139.3 A X 84.1 A, and the space group is I4. The electron density map at 6.5 A resolution shows that the enzyme is an octamer with D4 symmetry.

Crystallography↗
PubMed1983

Crystallization and 7 A resolution electron density map of the large fragment of Escherichia coli DNA polymerase I.

Crystals of the large fragment of Escherichia coli DNA polymerase I have been grown that diffract to better than 2.8 A resolution. They are in tetragonal space group P4(3) with a = b = 104.1 A, c = 86 A. A 7 A resolution map shows the protein to consist of two domains and to be mostly alpha-helical. The active site has been located by binding nucleoside monophosphates.

Binding Sites↗
PubMed1983

Crystallographic studies of protein-nucleic acid interaction: catabolite gene activator protein and the large fragment of DNA polymerase I.

Crystals suitable for X-ray crystallographic investigation have been grown of several nucleic acid binding proteins and their analysis is in progress. These include E. coli catabolite gene activator protein (CAP), the large fragment of DNA polymerase I (Pol I fragment), rec A, single strand DNA binding protein, resolvase, lac repressor and lac repressor 'Core', 5S RNA fragment and its complex with L25. Calculation of the electrostatic charge potential of CAP, using coordinates refined at 2.6 A resolution, suggests an orientation for B DNA on this repressor and activator of transcription. Both the electrostatic calculations and detailed model building suggests that the DNA must be bent or kinked on the protein in this orientation in order to make sufficient protein contacts. From a 3.5 A resolution map of Pol I fragment we have been able to obtain a preliminary trace through the polypeptide backbone. The large fragment consists of two domains. The smaller domain binds nucleoside monophosphate at the edge of a mostly parallel beta-pleated sheet, a structure that is reminiscent of kinase and dehydrogenase nucleotide binding domains. The larger domain contains about two thirds of the fragment and is mostly alpha-helical but with at least one four stranded antiparallel beta-sheet. The nucleoside monophosphate binds with its 5' phosphate on the Mg and is apparently in the conformation of nucleotides in B DNA.

Cyclic AMP Receptor Protein↗