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P Brick

Publications and source records attributed to P Brick.

At least 37 records · Page 2Linked to original sources
PubMed1995

The crystal structure of the lysyl-tRNA synthetase (LysU) from Escherichia coli.

BACKGROUND: Lysyl-tRNA synthetase catalyzes the attachment of the amino acid lysine to the cognate tRNA. The enzyme is a member of the class II amino-acyl-tRNA synthetases; the crystal structures of the seryl- and aspartyl-tRNA synthetases from this class are already known. Lysyl-tRNA synthetase shows extensive sequence homology with aspartyl-tRNA synthetase. In Escherichia coli there are two isoforms of the enzyme, LysS and LysU. Unlike LysS, which is synthesized under normal growth conditions, LysU is the product of a normally silent gene which is overexpressed under extreme physiological conditions (such as heat-shock), and can synthesize a number of adenyl dinucleotides (in particular AppppA). These dinucleotides have been proposed to act as modulators of the heat-shock response and stress response. RESULTS: The crystal structure of E. coli LysU has been determined to 2.8 A resolution, with lysine bound to the active site. The protein is a homodimer, with a rather extended dimer interface spanning the entire length of the molecule. Each monomer consists of two domains: a smaller N-terminal domain which binds the tRNA anticodon, and a larger C-terminal domain with the topology characteristic of the catalytic domain found in class II synthetases. CONCLUSIONS: A comparison of the LysU crystal structure with the structures of seryl- and aspartyl-tRNA synthetases enables a conserved core to be identified. The structural homology with the aspartyl-tRNA synthetase extends to include the anticodon-binding domain. When the active sites of lysyl-, aspartyl- and seryl-tRNA synthetases are compared, a number of catalytically important residues are conserved and a similar extended network of hydrogen bonds can be observed in the amino acid binding pocket in all three structures, although the details may differ. The lysine substrate is involved in an extended network of hydrogen bonds and polar interactions, with the side chain amino group forming a salt bridge with Glu428. The binding of ATP to LysU can be modelled on the basis of the aspartyl-tRNA synthetase-ATP complex, but the tRNA acceptor stem interaction for LysU cannot be easily modelled by similar extrapolation.

Amino Acid Sequence↗
PubMed1994

Crystallization and preliminary diffraction studies of Escherichia coli lysyl-tRNA synthetase (LysU).

Crystals of Escherichia coli lysyl-tRNA synthetase (lysU gene product) have been obtained by vapour diffusion techniques. Three different crystal forms could be grown under similar conditions. The crystals that have been chosen for the structure determination belong to space group C222(1) with cell dimensions a = 144.3 A, b = 257.8 A, c = 182.1 A and contain three monomers in the asymmetric unit. They diffract to at least 2.1 A resolution, but are very sensitive to radiation damage.

Crystallization↗
PubMed1994

Crystal structure of a NAD-dependent D-glycerate dehydrogenase at 2.4 A resolution.

D-Glycerate dehydrogenase (GDH) catalyzes the NADH-linked reduction of hydroxypyruvate to D-glycerate. GDH is a member of a family of NAD-dependent dehydrogenases that is characterized by a specificity for the D-isomer of the hydroxyacid substrate. The crystal structure of the apoenzyme form of GDH from Hyphomicrobium methylovorum has been determined by the method of isomorphous replacement and refined at 2.4 A resolution using a restrained least-squares method. The crystallographic R-factor is 19.4% for all 24,553 measured reflections between 10.0 and 2.4 A resolution. The GDH molecule is a symmetrical dimer composed of subunits of molecular mass 38,000, and shares significant structural homology with another NAD-dependent enzyme, formate dehydrogenase. The GDH subunit consists of two structurally similar domains that are approximately related to each other by 2-fold symmetry. The domains are separated by a deep cleft that forms the putative NAD and substrate binding sites. One of the domains has been identified as the NAD-binding domain based on its close structural similarity to the NAD-binding domains of other NAD-dependent dehydrogenases. The topology of the second domain is different from that found in the various catalytic domains of other dehydrogenases. A model of a ternary complex of GDH has been built in which putative catalytic residues are identified based on sequence homology between the D-isomer specific dehydrogenases. A structural comparison between GDH and L-lactate dehydrogenase indicates a convergence of active site residues and geometries for these two enzymes. The reactions catalyzed are chemically equivalent but of opposing stereospecificity. A hypothesis is presented to explain how the two enzymes may exploit the same coenzyme stereochemistry and a similar spatial arrangement of catalytic residues to carry out reactions that proceed to opposite enantiomers.

Amino Acid Sequence↗
PubMed1993

Crystal structure of cholesterol oxidase complexed with a steroid substrate: implications for flavin adenine dinucleotide dependent alcohol oxidases.

Cholesterol oxidase from Brevibacterium sterolicum is a flavin-dependent enzyme that catalyzes the oxidation and isomerization of 3 beta-hydroxy steroids with a double bond at delta 5-delta 6 of the steroid ring backbone. The crystal structure of the free enzyme in the absence of a steroid substrate has previously been determined. In this paper we report the crystal structure of the complex of cholesterol oxidase with the steroid substrate dehydroisoandrosterone, refined at 1.8-A resolution. The final crystallographic R-value is 15.7% for all reflections between 10.0- and 1.8-A resolution. The steroid is buried within the protein in an internal cavity which, in the free enzyme crystal structure, was occupied by a lattice of water molecules. The conformations of a number of side chains lining the active-site cavity have changed in order to accommodate the steroid substrate. A loop region of the structure between residues 70 and 90 differs significantly between the substrate-free and substrate-bound forms of the enzyme, presumably to facilitate binding of the steroid. The hydroxyl group of the steroid substrate is hydrogen-bonded to both the flavin ring system of the FAD cofactor and a bound water molecule. FAD-dependent cholesterol oxidase shares significant structural homology with another flavoenzyme, glucose oxidase, suggesting that it might also be a member of the glucose-methanol-choline (GMC) oxidoreductase family. Although there is only limited sequence homology, a superposition of these two structures reveals a conserved histidine residue within hydrogen-bonding distance of the active-site water molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Brevibacterium↗
PubMed1992

Crystallization and preliminary diffraction studies of hydroxypyruvate reductase (D-glycerate dehydrogenase) from Hyphomicrobium methylovorum.

Two crystal forms of hydroxypyruvate reductase (D-glycerate dehydrogenase) from the methylotrophic bacterium Hyphomicrobium methylovorum have been grown from ammonium sulphate solutions. One crystal form is triclinic, with unit cell parameters a = 60.4 A, b = 60.5 A, c = 66.3 A, alpha = 102.3 degrees, beta = 113.7 degrees and gamma = 102.7 degrees, suggesting that a dimer (monomer M(r) 38,000) occupies the unit cell. This crystal form diffracts to beyond 2.4 A resolution and is suitable for crystallographic structure analysis.

Alcohol Oxidoreductases↗
PubMed1991

Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina caffra seeds.

The trypsin inhibitor DE-3 from Erythrina caffra (ETI) belongs to the Kunitz-type soybean trypsin inhibitor (STI) family and consists of 172 amino acid residues with two disulphide bridges. The amino acid sequence of ETI shows high homology to other trypsin inhibitors from the same family but ETI has the unique ability to bind and inhibit tissue plasminogen activator. The crystal structure of ETI has been determined using the method of isomorphous replacement and refined using a combination of simulated annealing and conventional restrained least-squares crystallographic refinement. The refined model includes 60 water molecules and 166 amino acid residues, with a root-mean-square deviation in bond lengths from ideal values of 0.016 A. The crystallographic R-factor is 20.8% for 7770 independent reflections between 10.0 and 2.5 A. The three-dimensional structure of ETI consists of 12 antiparallel beta-strands joined by long loops. Six of the strands form a short antiparallel beta-barrel that is closed at one end by a "lid" consisting of the other six strands coupled in pairs. The molecule shows approximate 3-fold symmetry about the axis of the barrel, with the repeating unit consisting of four sequential beta-strands and the connecting loops. Although there is no sequence homology, this same fold is present in the structure of interleukin-1 alpha and interleukin-1 beta. When the structure of ETI and interleukin-1 beta are superposed, the close agreement between the alpha-carbon positions for the beta-strands is striking. The scissile bond (Arg63-Ser64) is located on an external loop that protrudes from the surface of the molecule and whose architecture is not constrained by secondary structure elements, disulphide bridges or strong electrostatic interactions. The hydrogen bonds made by the side-chain amide group of Asn12 play a key role in maintaining the three-dimensional structure of the loop. This residue is in a position corresponding to that of a conserved asparagine in the Kazal inhibitor family. Although the overall structure of ETI is similar to the partial structure of STI, the scissile bond loop is displaced by about 4 A. This displacement probably arises from the fact that the structure of STI has been determined in a complex with trypsin but could possibly be a consequence of the close molecular contact between Arg63 and an adjacent molecule in the crystal lattice.

Amino Acid Sequence↗
PubMed1989

Crystallization and preliminary diffraction studies of Erythrina trypsin inhibitor.

Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.

Crystallization↗
PubMed1989

Structure of tyrosyl-tRNA synthetase refined at 2.3 A resolution. Interaction of the enzyme with the tyrosyl adenylate intermediate.

The crystal structure of tyrosyl-tRNA synthetase (EC 6.1.1.1) from Bacillus stearothermophilus has been refined to a crystallographic R-factor of 22.6% at 2.3 A resolution using a restrained least-squares procedure. In the final model the root-mean-square deviation from ideality for bond distances is 0.018 A and for angle distances is 0.044 A. Each monomer consists of three domains: an alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet, an alpha-helical domain (248 to 318) containing five helices, and a disordered C-terminal domain (319 to 418) for which the electron density is very weak and where it has not been possible to trace the polypeptide chain. Complexes of the enzyme with the catalytic intermediate tyrosyl adenylate and the inhibitor tyrosinyl adenylate have also been refined to R-factors of 23.9% at 2.8 A resolution and 21.0% at 2.7 A resolution, respectively. Formation of the complexes results in some crystal cracking, but there is no significant difference in the conformation of the polypeptide chain of the three structures described here. The relative orientation of the alpha/beta and alpha-helical domains is similar to that previously observed for the "A" subunit of a deletion mutant lacking the C-terminal domain. Differences between these structures are confined to surface loops that are involved in crystal packing. Tyrosyl adenylate and tyrosinyl adenylate bind in similar conformations within a deep cleft in the alpha/beta domain. The tyrosine moiety is in the equivalent position to that occupied by tyrosine in crystals of the truncated mutant and makes similar strong polar interactions with the enzyme. The alpha-phosphate group interacts with the main-chain nitrogen of Asp38. The two hydroxyl groups of the ribose form strong interactions with the protein. The 2'-hydroxyl group interacts with the carboxylate of Asp194 and the main-chain nitrogen of Gly192 while the 3'-hydroxyl interacts with a tightly bound water molecule (Wat326). The adenine moiety appears to make no significant polar interactions with the protein. The results of site-directed mutagenesis studies are examined in the light of these refined structures.

Adenine↗
PubMed1987

Crystal structure of a deletion mutant of a tyrosyl-tRNA synthetase complexed with tyrosine.

The crystal structure of a deletion mutant of tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been determined at 2.5 A resolution using molecular replacement techniques. The genetically engineered molecule catalyses the activation of tyrosine with kinetic properties similar to those of the wild-type enzyme but no longer binds tRNATyr. It contains 319 residues corresponding to the region of the polypeptide chain for which interpretable electron density is present in crystals of the wild-type enzyme. The partly refined model of the wild-type enzyme was used as a starting point in determining the structure of the truncated mutant. The new crystals are of space group P2(1) and contain the molecular dimer within the asymmetric unit. The refined model has a crystallographic R-factor of 18.7% for all reflections between 8 and 2.5 A. Each subunit contains two structural domains: the alpha/beta domain (residues 1 to 220) containing a six-stranded beta-sheet and the alpha-helical domain (residues 248 to 319) containing five helices. The alpha/beta domains are related by a non-crystallographic dyad while the alpha-helical domains are in slightly different orientations in the two subunits. The tyrosine substrate binds in a slot at the bottom of a deep active site cleft in the middle of the alpha/beta domain. It is surrounded by polar side-chains and water molecules that are involved in an intricate hydrogen bonding network. Both the alpha-amino and hydroxyl groups of the substrate make good hydrogen bonds with the protein. The amino group forms hydrogen bonds with Tyr169-OH, Asp78-OD1 and Gln173-OE1. The phenolic hydroxyl group forms hydrogen bonds with Asp76-OD1 and Tyr34-OH. In contrast, the substrate carboxyl group makes no direct interactions with the enzyme. The results of both substrate inhibition studies and site-directed mutagenesis experiments have been examined in the light of the refined structure.

Amino Acid Sequence↗
PubMed1984

Interaction of crystalline tyrosyl-tRNA synthetase with adenosine, adenosine monophosphate, adenosine triphosphate and pyrophosphate in the presence of tyrosinol.

Crystalline complexes of tyrosyl-tRNA synthetase from Bacillus stearothermophilus were prepared with adenosine, AMP, ATP and PPi, all in the presence of tyrosinol, which binds strongly to the tyrosine binding site but cannot be adenylated by ATP. The hydrolysis of ATP in the presence of crystalline tyrosyl-tRNA synthetase (or redissolved crystals) was checked in the absence of tyrosine or with tyrosinol. No ATPase activity due to the enzyme was detected under these conditions. Difference Fourier analysis shows that tyrosinol binds to the tyrosine binding site with the same occupancy as the amino acid. Comparison between tyrosine and tyrosinol shows the location of the extra oxygen atom of the tyrosine carboxylate. Adenosine, AMP and ATP are weakly bound to the enzyme in the presence of tyrosinol. Even when ATP is present at a concentration greater than Km for adenylation, it is not sufficiently strongly bound to give a recognizable density for adenine. However, some significant peaks of density are present near the tyrosine binding site. One of them is at the usual ribose binding site, and may possibly represent ribose binding with a low occupancy. When AMP is bound a similar but not identical arrangement of density is observed.

Adenosine↗
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↗