Biomedical subjects

Louis T J Delbaere

Publications and source records attributed to Louis T J Delbaere.

13 recordsLinked to original sources

PubMed2006

The structure of an ancient conserved domain establishes a structural basis for stable histidine phosphorylation and identifies a new family of adenosine-specific kinases.

Phosphorylation of both small molecules and proteins plays a central role in many biological processes. In proteins, phosphorylation most commonly targets the oxygen atoms of Ser, Thr, and Tyr. In contrast, stably phosphorylated His residues are rarely found, due to the lability of the N-P bond, and histidine phosphorylation features most often in transient processes. Here we present the crystal structure of a protein of previously unknown function, which proves to contain a stably phosphorylated histidine residue. The protein is the product of open reading frame PAE2307, from the hyperthermophilic archaeon Pyrobaculum aerophilum, and is representative of a highly conserved protein family found in archaea and bacteria. The crystal structure of PAE2307, solved at 1.45-A resolution (R = 0.208, R(free) = 0.227), forms a remarkably tightly associated hexamer. The phosphorylated histidine at the proposed active site, pHis85, occupies a cavity that is at the interface between two subunits and contains a number of fully conserved residues. Stable phosphorylation is attributed to favorable hydrogen bonding of the phosphoryl group and a salt bridge with pHis85 that provides electronic stabilization. In silico modeling suggested that the protein may function as an adenosine kinase, a conclusion that is supported by in vitro assays of adenosine binding, using fluorescence spectroscopy, and crystallographic visualization of an adenosine complex of PAE2307 at 2.25-A resolution.

Adenosine↗

PubMed2006

Substrate-free structure of a monomeric NADP isocitrate dehydrogenase: an open conformation phylogenetic relationship of isocitrate dehydrogenase.

Both monomeric and dimeric NADP+-dependent isocitrate dehydrogenase (IDH) belong to the metal-dependent beta-decarboxylating dehydrogenase family and catalyze the oxidative decarboxylation from 2R,3S-isocitrate to yield 2-oxoglutarate, CO2, and NADPH. It is important to solve the structures of IDHs from various species to correlate with its function and evolutionary significance. So far, only two crystal structures of substrate/cofactor-bound (isocitrate/NADP) NADP+-dependent monomeric IDH from Azotobacter vinelandii (AvIDH) have been solved. Herein, we report for the first time the substrate/cofactor-free structure of a monomeric NADP+-dependent IDH from Corynebacterium glutamicum (CgIDH) in the presence of Mg2+. The 1.75 A structure of CgIDH-Mg2+ showed a distinct open conformation in contrast to the closed conformation of AvIDH-isocitrate/NADP+ complexes. Fluorescence studies on CgIDH in the presence of isocitrate/or NADP+ suggest the presence of low energy barrier conformers. In CgIDH, the amino acid residues corresponding to the Escherichia coli IDH phosphorylation-loop are alpha-helical compared with the more flexible random-coil region in the E. coli protein where IDH activation is controlled by phosphorylation. This more structured region supports the idea that activation of CgIDH is not controlled by phosphorylation. Monomeric NADP+-specific IDHs have been identified from about 50 different bacterial species, such as proteobacteria, actinobacteria, and planctomycetes, whereas, dimeric NADP+-dependent IDHs are diversified in both prokaryotes and eukaryotes. We have constructed a phylogenetic tree based on amino acid sequences of all bacterial monomeric NADP+-dependent IDHs and also another one with specifically chosen species which either contains both monomeric and dimeric NADP+-dependent IDHs or have monomeric NADP+-dependent, as well as NAD+-dependent IDHs. This is done to examine evolutionary relationships.

Bacterial Proteins↗

PubMed2005

Crystallization, preliminary X-ray diffraction and structure solution of MosA, a dihydrodipicolinate synthase from Sinorhizobium meliloti L5-30.

The structure of MosA, a dihydrodipicolinate synthase and reported methyltransferase from Sinorhizobium meliloti, has been solved using molecular replacement with Escherichia coli dihydrodipicolinate synthase as the model. A crystal grown in the presence of pyruvate diffracted X-rays to 2.3 A resolution using synchrotron radiation and belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 69.14, b = 138.87, c = 124.13 A.

Bacterial Proteins↗

PubMed2005

Structure of PEP carboxykinase from the succinate-producing Actinobacillus succinogenes: a new conserved active-site motif.

Actinobacillus succinogenes can produce, via fermentation, high concentrations of succinate, an important industrial commodity. A key enzyme in this pathway is phosphoenolpyruvate carboxykinase (PCK), which catalyzes the production of oxaloacetate from phosphoenolpyruvate and carbon dioxide, with the concomitant conversion of adenosine 5'-diphosphate to adenosine 5'-triphosphate. 1.85 and 1.70 A resolution structures of the native and a pyruvate/Mn(2+)/phosphate complex have been solved, respectively. The structure of the complex contains sulfhydryl reducing agents covalently bound to three cysteine residues via disulfide bonds. One of these cysteine residues (Cys285) is located in the active-site cleft and may be analogous to the putative reactive cysteine of PCK from Trypanosoma cruzi. Cys285 is also part of a previously unreported conserved motif comprising residues 280-287 and containing the pattern NXEXGXY(/F)A(/G); this new motif appears to have a structural role in stabilizing and positioning side chains that bind substrates and metal ions. The first few residues of this motif connect the two domains of the enzyme and a fulcrum point appears to be located near Asn280. In addition, an active-site Asp residue forms two coordinate bonds with the Mn(2+) ion present in the structure of the complex in a symmetrical bidentate manner, unlike in other PCK structures that contain a manganese ion.

Actinobacillus↗

PubMed2005

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop.

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.4 Angstroms resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383-397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel beta-sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site.

Adenosine Triphosphate↗

PubMed2004

Structure/function studies of phosphoryl transfer by phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (PCK) catalyzes the conversion of oxaloacetate (OAA) to PEP and carbon dioxide with the subsequent conversion of nucleoside triphosphate to nucleoside diphosphate (NDP). The 1.9 A resolution structure of Escherichia coli PCK consisted of a 275-residue N-terminal domain and a 265-residue C-terminal domain with the active site located in a cleft between these domains. Each domain has an alpha/beta topology and the overall structure represents a new protein fold. Furthermore, PCK has a unique mononucleotide-binding fold. The 1.8 A resolution structure of the complex of ATP/Mg(2+)/oxalate with PCK revealed a 20 degrees hinge-like rotation of the N- and C-terminal domains, which closed the active site cleft. The ATP was found in the unusual syn conformation as a result of binding to the enzyme. Along with the side chain of Lys254, Mg(2+) neutralizes charges on the P beta and P gamma oxygen atoms of ATP and stabilizes an extended, eclipsed conformation of the P beta and P gamma phosphoryl groups. The sterically strained high-energy conformation likely lowers the free energy of activation for phosphoryl transfer. Additionally, the gamma-phosphoryl group becomes oriented in-line with the appropriate enolate oxygen atom, which strongly supports a direct S(N)2-type displacement of this gamma-phosphoryl group by the enolate anion. In the 2.0 A resolution structure of the complex of PCK/ADP/Mg(2+)/AlF(3), the AlF(3) moiety represents the phosphoryl group being transferred during catalysis. There are three positively charged groups that interact with the fluorine atoms, which are complementary to the three negative charges that would occur for an associative transition state.

Adenosine Diphosphate↗

PubMed2004

The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus.

V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

Aspartic Acid↗

PubMed2003

Crystallization and preliminary X-ray crystallographic studies of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum.

Phosphoenolpyruvate carboxykinase (PCK) is a key enzyme involved in the regulation of gluconeogenesis. PCKs from higher animals require guanosine nucleotide for activity. PCK from Corynebacterium glutamicum is also GTP specific. X-ray diffraction data from a C. glutamicum PCK crystal were collected to 2.8 A resolution. The crystals were monoclinic, belonging to space group P2(1), with unit-cell parameters a = 71.7, b = 117.4, c = 161.3 A, beta = 92.9 degrees. The presence of two molecules in the crystallographic asymmetric unit gives a V(M) of 2.5 A(3) Da(-1) and a solvent content of 50.3%.

Actinomycetales↗

PubMed2003

Cobalt(II), nickel(II) and zinc(II) do not bind to intra-helical N(7) guanine positions in the B-form crystal structure of d(GGCGCC).

Three novel X-ray crystal structures for the DNA hexamer d(GGCGCC) in the B-form complexed to divalent cobalt, nickel and zinc ions have been determined to a resolution of 2.9-3.0 A. The structures were isomorphous and had five DNA strands and five metal cations per asymmetric unit. In all three cases, divalent metal cations were coordinated only to the terminal guanine residue at the N(7) position, with no metal ions binding to non-terminal guanine positions. Water molecules bound to the metal cations interacted with neighboring guanine residues 3' to the ones to which the cations were coordinated, affecting the propeller twist. Even though DNA occupied only about 35% of the unit cell volume, it is interesting that the few interactions involving the metal cations were sufficient to stabilize the crystal lattice. As well as lending support to the proposal that these metals do not coordinate to B-DNA in a stable manner, the results presented here also extend the crystallographic evidence for this phenomenon to the GGC and CGC sequences for all three metal cations.

Binding Sites↗

PubMed2003

Expression, purification, and characterization of a bacterial GTP-dependent PEP carboxykinase.

The Corynebacterium glutamicum (C. glutamicum) phosphoenolpyruvate carboxykinase (PCK) gene (pckA) was cloned into an Escherichia coli expression vector with a glutathione S-transferase (GST) tag. This recombinant DNA can produce highly overexpressed tagged protein in soluble form. This is the first report of the production of C. glutamicum PCK overexpressed in E. coli. The GST-fused PCK was purified using the glutathione-Sepharose 4B affinity column and the GST tag was removed in one-step. This one-step, easy purification method would be very useful for future mutational and structural studies. The molecular mass of the purified protein is approximately 68 kDa as confirmed by mass spectrometry and it is a monomeric enzyme. Also, the enzyme assays revealed that C. glutamicum PCK has a GTP-specific activity and that its activity is maximal in the presence of both Mn2+ and Mg2+.

Amino Acid Sequence↗

PubMed2003

Mechanisms of activation of phosphoenolpyruvate carboxykinase from Escherichia coli by Ca2+ and of desensitization by trypsin.

The 1.8-A resolution structure of the ATP-Mg(2+)-Ca(2+)-pyruvate quinary complex of Escherichia coli phosphoenolpyruvate carboxykinase (PCK) is isomorphous to the published complex ATP-Mg(2+)-Mn(2+)-pyruvate-PCK, except for the Ca(2+) and Mn(2+) binding sites. Ca(2+) was formerly implicated as a possible allosteric regulator of PCK, binding at the active site and at a surface activating site (Glu508 and Glu511). This report found that Ca(2+) bound only at the active site, indicating that there is likely no surface allosteric site. (45)Ca(2+) bound to PCK with a K(d) of 85 micro M and n of 0.92. Glu508Gln Glu511Gln mutant PCK had normal activation by Ca(2+). Separate roles of Mg(2+), which binds the nucleotide, and Ca(2+), which bridges the nucleotide and the anionic substrate, are implied, and the catalytic mechanism of PCK is better explained by studies of the Ca(2+)-bound structure. Partial trypsin digestion abolishes Ca(2+) activation (desensitizes PCK). N-terminal sequencing identified sensitive sites, i.e., Arg2 and Arg396. Arg2Ser, Arg396Ser, and Arg2Ser Arg396Ser (double mutant) PCKs altered the kinetics of desensitization. C-terminal residues 397 to 540 were removed by trypsin when wild-type PCK was completely desensitized. Phe409 and Phe413 interact with residues in the Ca(2+) binding site, probably stabilizing the C terminus. Phe409Ala, DeltaPhe409, Phe413Ala, Delta397-521 (deletion of residues 397 to 521), Arg396(TAA) (stop codon), and Asp269Glu (Ca(2+) site) mutations failed to desensitize PCK and, with the exception of Phe409Ala, appeared to have defects in the synthesis or assembly of PCK, suggesting that the structure of the C-terminal domain is important in these processes.

Binding Sites↗

PubMed2003

Mapping protein: carbohydrate interactions.

Many biologically important interactions occur between proteins and carbohydrates. The examination of these interactions at the atomic level is critical not only in understanding the nature of these interactions and their biological role, but also in the design of effective modulators of these interactions. While experimentally obtained structural information is preferred, quite often this information is unavailable. In order to address this, several methods have been developed to probe the interactions between protein and carbohydrate in the absence of structural data. These methods map the interactions between protein and carbohydrate, and identify the groups involved, both at the carbohydrate and protein level. Here, we review these developments, and examine the strengths, weaknesses, and pitfalls of these methods.

Animals↗

PubMed2002

Jel44 monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and the complex of Jel44 Fab fragment with HPr: preparation, crystallization and preliminary crystallographic analysis.

Jel44 is a mouse monoclonal antibody specific for the histidine-containing phosphocarrier protein (HPr), a component of a sugar-transport system in Escherichia coli. Because Jel44 binding to HPr is dependent upon ionic strength and the enthalpic and entropic contributions do not vary over the temperature range 277-310 K, the complex is of great interest. A single crystal of the Jel44 Fab fragment was obtained and diffracted X-rays to a maximum resolution of 4.6 A on an in-house X-ray source. The crystal belongs to space group P2(1), with unit-cell parameters a = 68.6, b = 67.7, c = 105.5 A, beta = 96 degrees. Although crystals of the complex of Jel44 Fab fragment with HPr could not be fully characterized owing to suspected crystal twinning, it was encouraging that they diffracted X-rays to 2.5 A on an in-house X-ray source. It is thus foreseen that improvement of crystal quality will allow the complete solution of this novel structure.

Animals↗