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At least 811 records · Page 45Linked to original sources
PubMed2006

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

Calorimetry, Differential Scanning↗
PubMed2006

Role of native-state structure in rubredoxin native-state hydrogen exchange.

Amide exchange rates were measured for Pyrococcus furiosus (Pf) rubredoxin substituted with either Zn(II), Ga(III), or Ge(IV). Base-catalyzed exchange rate constants increase up to 3000-fold per unit charge for the highly protected amides surrounding the active site metal, yielding apparent residue-specific conformational energy decreases of more than 8 kcal/mol in a comparison of the Zn(II)- and Ge(IV)-substituted proteins. However, the exchange kinetics for many of the other amides of the protein are insensitive to these metal substitutions. These differential rates are inversely correlated with the distance between the amide nitrogen and the metal in the X-ray structure, out to a distance of at least 12 A, consistent with an electrostatic potential-dependent shifting of the amide nitrogen pK. This strongly correlated distance dependence is consistent with a nativelike structure for the exchange-competent conformations. The electric field potential within the interior of the rubredoxin structure gives rise to a change of as much as a million-fold in the rate for the exchange-competent state of the individual amide hydrogens. Nevertheless, the strength of these electrostatic interactions in Pf rubredoxin appears to be comparable to those previously reported within other proteins. As a result, contrary to the conventional analysis of hydrogen exchange data, for exchange processes that occur via nonglobal transitions, the residual conformational structure will often modulate the observed rates. Although this necessarily complicates the estimation of the conformational equilibria of these exchange-competent states, this dependence on residual structure can provide insight into the conformation of these transient states.

Amides↗
PubMed1998

Kinetic role of electrostatic interactions in the unfolding of hyperthermophilic and mesophilic rubredoxins.

The temperature dependence of the unfolding kinetics of rubredoxins from the hyperthermophile Pyrococcus furiosus (RdPf) and the mesophile Clostridium pasteurianum (RdCp) has been studied. Results show that RdPf unfolds much more slowly, under all experimentally accessible temperature regimes, than RdCp and other typical mesophilic proteins. Rates of RdCp and RdPf unfolding decrease upon increasing the pH above 2 and diverge dramatically at pH 7. As shown by detailed electrostatic energy calculations, this is the result of a differential degree of protonation of the negatively charged amino acids, which causes distinct electrostatic configurations as a function of pH. We propose that ion pairs, particularly those that are placed in key surface positions, may play a kinetic role by mildly clamping the protein and thereby influencing the nature and the number of the vibrational normal modes that are thermally accessible upon unfolding. More generally, these modes are also likely to be affected by the favorable electrostatic configurations, which we have shown to be directly linked to the extremely slow unfolding rates of RdPf at neutral pH. Even at pH 2, in the absence of any salt bridges, the unfolding rates of RdPf are much smaller than those of RdCp. This is ascribed to presently unidentified structural elements of nonelectrostatic nature. Since electrostatic effects influence the unfolding kinetics of both mesophilic and thermophilic rubredoxins, these findings may be of general significance for proteins.

Archaeal Proteins↗
PubMed1998

Thermodynamic stability of archaeal histones.

The temperature, salt, and pH dependencies of unfolding of four recombinant (r) archaeal histones (rHFoB from the mesophile Methanobacterium formicicum, and rHMfA, rHMfB, and rHPyA1 from the hyperthermophiles Methanothermus fervidus and Pyrococcus strain GB-3a) have been determined by circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). The thermal unfolding of these proteins is > 90% reversible, with concentration-dependent apparent Tm values and asymmetric unfolding transitions that are fit well by a two-state unfolding model in which a histone dimer unfolds to two random coil monomers. rHPyA1 dimers are stable in the absence of salt, whereas rHMfA, rHMfB, and rHFoB dimers unfold at 20 degrees C and pH 2 in solutions containing < 200 mM, < 400 mM, and < 1.5 M KCl, respectively. rHMfA, rHMfB, and rHFoB also experience significant cold denaturation in low salt concentrations and at low pH. The midpoint of thermal unfolding of a 1 M protein solution (T degree value) and the temperature dependency of the free energy of unfolding have been established for each histone, and both parameters correlate with the growth temperature of the originating archaeon. The changes in heat capacity upon unfolding are similar for the four histones, indicating that enhanced thermostability is not obtained by altering the curvature of the stability curve. Rather, the stability curves for the histones from the hyperthermophiles are displaced vertically to higher energies and laterally to higher Tmax values relative to the stability curve for rHFoB. The maximal free energies of unfolding for rHFoB, rHMfA, rHMfB, and rHPyA1 are 7.2, 15.5, 14.6, and 17.2 kcal/mol at 32, 35, 40, and 44 degrees C, respectively. T degree values for rHFoB, rHMfA, rHMfB, and rHPyA1 are 75, 104, 113, and 114 degrees C, respectively, at pH 5 in 0.2 M KCl. Structural features within the conserved histone fold that might confer these stability differences are discussed.

Amino Acid Sequence↗
PubMed1999

Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature.

The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature. Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C. The reduction potential of P. furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm. Thus, the reduction potential of P. furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions. Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C. pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P. furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles. The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology. Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity. The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI). A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations. A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.

Clostridium↗
PubMedDate not supplied

Strategic selection of hyperthermophilic esterases for resolution of 2-arylpropionic esters.

Homologues to Carboxylesterase NP and Candida rugosa lipase, used for the chiral separation of racemic mixtures of 2-arylpropionic methyl esters, were identified by BLAST searches of available genome sequences for hyperthermophilic microorganisms. Two potential candidates were identified: a putative lysophospholipase from Pyrococcus furiosus (Pfu-LPL) and a carboxylesterase from Sulfolobus solfataricus P1 (Sso-EST1). Although both enzymes showed hydrolytic preference toward the (S) methyl ester, only Sso-EST1 yielded highly optically pure (S) naproxen (%ee(p) >/= 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %ee(p) or E values with Sso-EST1. However, the addition of 25% methanol resulted in a 25% increase in E. The effect of various cosolvents on the enantiomeric ratio showed no correlation with the log P or dielectric constant values of the solvent. However, an inverse relationship between E and the denaturation capacity (DC) of the water miscible cosolvents was observed. This was attributed to an increase in enzyme flexibility with increasing solvent DC values leading to a concomitant reduction in the resolving power of Sso-EST1. The results here show that although bioinformatics tools can be used to select candidate biocatalysts for chiral resolution of 2-arylpropionic esters, biochemical characterization is needed to definitively determine functional characteristics.

Amino Acid Sequence↗
PubMed2002

O2 penetration and proton burial depth in proteins: applicability to fold family recognition.

Paramagnetically induced relaxation effects of O2 and the nitroxide 4-hydroxy TEMPO were measured for the amide protons of perdeuterated rubredoxin from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium pasteurianum. For both O2 and the impermeant nitroxide, the induced relaxation at the static solvent inaccessible amide sites is dominated by long-range interactions with the paramagnetic species in the bulk aqueous phase. The upper bound of O2 solubility in the internal matrix of the rubredoxins is one-tenth that of the bulk aqueous phase. Furthermore, the difference between the oxygen solubilities inside the two rubredoxins is at most 1% that of bulk water O2 solubility, suggesting that there are only modest differences in this measure of fluidity for the mesophile vs hyperthermophile protein interiors. Calculations based on the assumption of a paramagnet uniformly distributed on the protein exterior yield accurate predictions at nearly all amide sites for the minimum relaxation value observed from either the O2 or nitroxide data. Model calculations indicate that the readily obtained paramagnetically induced relaxation effects should prove effective in recognition of structural homology for proteins that are too widely diverged for sequence-based recognition.

Bacterial Proteins↗
PubMed2003

Discovery of the archaeal chemical link between glycogen (starch) synthase families using a new mass spectrometry assay.

Starch and its analogue glycogen are biosynthesized by enzymes that have been classified by sequence similarities into two families that have no significant sequence overlap: the animal/fungal glycogen synthases and the plant/bacterial glycogen (starch) synthases. Recent gene sequence analysis of putative archaea enzymes implicates them as a third family that links the structural and functional features of the other two classes. Herein, we present the first rapid electrospray ionization mass spectrometry-based assay to quantify any carbohydrate-polymerizing activity, the first cloning and recombinant expression as well as verification of the putative function of a glycogen synthase from the hyperthermophilic archaea Pyrococcus furiosus, and the characterization of a variety of glycogen synthases with the new assay. The new assay allowed the determination of Km and Vmax values for the rabbit, yeast, and P. furiosus glycogen synthases. Most surprisingly, unlike the synthases from rabbit or yeast and in contradiction to what would be expected from structural studies of other nucleotide-sugar binding proteins, the synthase from the archaea source accepts both uridine- and adenine-diphosphate activated glucose competitively and with comparable affinities to form a glucose polymer. This loose substrate specificity implicates this protein as the chemical link between the two branches of glycogen synthases that have evolved to accept primarily one or the other nucleotide as well as a good source enzyme for polymer bioengineering efforts.

Adenosine Diphosphate Glucose↗
PubMed2005

One-step synthesis of labeled sugar nucleotides for protein O-GlcNAc modification studies by chemical function analysis of an archaeal protein.

Herein we present the chemical function analysis of a recombinant sugar nucleotidyltransferase from the hyperthermophile Pyrococcus furiosus and its use in the one-pot synthesis of chloroacetyl- and alkyne-tagged analogues of uridinediphospho-N-acetylglucosamine (UDP-GlcNAc). The gene was originally annotated as a glucose-1-phosphate deoxythymidylyltransferase; however, kinetic analysis of a panel of sugar-1-phosphates with the protein shows that it is better described as a bifunctional protein that synthesizes UDP-GlcNAc from glucosamine-1-phosphate and acetyl coenzyme A (CoA). A new mass-spectrometry-based assay for the rapid analysis of the acyltransferase activity demonstrates that the enzyme can also accept cheaper truncated N-acetylcysteamine thioester substrates in place of the natural acetyl CoA. The enzyme can tolerate alkyne or chloride substitutions in the acyl moiety, thereby allowing the facile synthesis of tagged sugar nucleotides for future use in protein O-GlcNAc modification studies.

Archaeal Proteins↗
PubMed2004

Unusually broad substrate tolerance of a heat-stable archaeal sugar nucleotidyltransferase for the synthesis of sugar nucleotides.

Herein, we report the first cloning, recombinant expression, and synthetic utility of a sugar nucleotidyltransferase from any archaeal source and demonstrate by an electrospray ionization mass spectrometry (ESI-MS)-based assay its unusual tolerance of heat, pH, and sugar substrates. The metal-ion-dependent enzyme from Pyrococcus furiosus DSM 3638 showed a relatively high degree of acceptance of glucose-1-phosphate (Glc1P), mannose-1-phosphate (Man1P), galactose-1-phosphate (Gal1P), fucose-1-phosphate, glucosamine-1-phosphate, galactosamine-1-phosphate, and N-acetylglucosamine-1-phosphate with uridine and deoxythymidine triphosphate (UTP and dTTP, respectively). The apparent Michaelis constants for Glc1P, Man1P, and Gal1P are 13.0 +/- 0.7, 15 +/- 1, and 22 +/- 2 microM, respectively, with corresponding turnover numbers of 2.08, 1.65, and 1.32 s(-1), respectively. An initial velocity study indicated an ordered bi-bi catalytic mechanism for this enzyme. The temperature stability and inherently broad substrate tolerance of this archaeal enzyme promise an effective reagent for the rapid chemoenzymatic synthesis of a range of natural and unnatural sugar nucleotides for in vitro glycosylation studies and highlight the potential of archaea as a source of new enzymes for synthesis.

Amino Acid Sequence↗
PubMed2006

Farnesyl diphosphate synthase: the art of compromise between substrate selectivity and stereoselectivity.

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.

Artemisia↗
PubMed2004

Enzymatic production and complete nuclear magnetic resonance assignment of the sugar lactulose.

The enzymatic transgalactosylation from lactose to fructose leading to the prebiotic disaccharide lactulose was investigated using the beta-galactosidase from Aspergillus oryzae and the hyperthermostable beta-glycosidase from Pyrococcus furiosus (CelB). The conditions for highest lactulose yields relative to the initial lactose concentration were established on a 1 mL scale. Dependent on the initial molar ratio of lactose to fructose, more or fewer oligosaccharides other than lactulose were generated. Bioconversions on a 30 mL scale in a stirred glass reactor were performed, and lactulose yields of 46 mmol/L (44% relative to lactose) for CelB and 30 mmol/L (30% relative to lactose) for A. oryzae beta-galactosidase were achieved. Only <5% of other oligosaccharides were detectable. The corresponding productivities were 24 and 16 mmol/L/h, respectively. The molecular structure of lactulose was investigated in detail and confirmed after purification of the reaction solution by LC-MS and 1D and 2D NMR. Lactulose (4-O-beta-D-galactopyranosyl-D-fructose) was unambiguously proved to be the major transglycosylation disaccharide.

Aspergillus oryzae↗
PubMed1999

Quantitative analysis of pyroglutamic acid in peptides.

A simplified and rapid procedure for the determination of pyroglutamic acid in peptides was developed. The method involves the enzymatic cleavage of an N-terminal pyroglutamate residue using a thermostable pyroglutamate aminopeptidase and isocratic HPLC separation of the resulting enzymatic hydrolysate using a column switching technique. Pyroglutamate aminopeptidase from a thermophilic archaebacteria, Pyrococcus furiosus, cleaves N-terminal pyroglutamic acid residue independent of the molecular weight of the substrate. It cleaves more than 85% of pyroglutamate from peptides whose molecular weight ranges from 362.4 to 4599.4 Da. Thus, a new method is presented that quantitatively estimates N-terminal pyroglutamic acid residue in peptides.

Amphibian Proteins↗
PubMed1998

Novel disulfide oxidoreductase in search of a function.

The high-resolution structure of an oxidoreductase from the archaeon Pyrococcus furiosus shows that it comprises two adjacent domains each with the thioredoxin (trx/grx) fold. Its functional properties are not yet fully defined but it may be related to the multi-domain eukaryotic protein disulfide-isomerases.

Binding Sites↗
PubMed2000

The orientation of DNA in an archaeal transcription initiation complex.

RNA polymerase from the hyperthermophile archaeon Pyrococcus furiosus (Pfu) forms specific and transcriptionally active complexes with its conjugate transcription factors TBP (the archaeal TATA binding protein homolog) and TFB (the archaeal homolog of eukaryotic RNA polymerase II and III transcription factors TFIIB and Brf) at the Pfu glutamate dehydrogenase promoter. A photochemical crosslinking method was used to map the vicinity of the catalytic subunits of Pfu RNA polymerase to DNA locations distributed along the polymerase-promoter interface. The largest component of this archaeal polymerase is split into two subunits, A' and A", whose relatively sharp boundary of DNA crosslinking (probed on the transcribed strand) is centered five to six base pairs downstream of the transcriptional start site. A strong argument based on this information, on the well-defined homology between the core bacterial, archaeal and eukaryotic RNA polymerase subunits, and on the recently determined structure of a bacterial RNA polymerase specifies the directionality of DNA in the archaeal transcription complex and its trajectory downstream of the transcriptional start site.

Archaeal Proteins↗
PubMed2001

Crystal structure of a DNA-dependent RNA polymerase (DNA primase).

Primases are essential components of the DNA replication apparatus in every organism. They catalyze the synthesis of oligoribonucleotides on single-stranded DNA, which subsequently serve as primers for the replicative DNA polymerases. In contrast to bacterial primases, the archaeal enzymes are closely related to their eukaryotic counterparts. We have solved the crystal structure of the catalytic primase subunit from the hyperthermophilic archaeon Pyrococcus furiosus at 2.3 A resolution by multiwavelength anomalous dispersion methods. The structure shows a two-domain arrangement with a novel zinc knuckle motif located in the primase (prim) domain. In this first structure of a complete protein of the archaeal/eukaryotic primase family, the arrangement of the catalytically active residues resembles the active sites of various DNA polymerases that are unrelated in fold.

Amino Acid Motifs↗
PubMed2003

Crystal structure of the human angiotensin-converting enzyme-lisinopril complex.

Angiotensin-converting enzyme (ACE) has a critical role in cardiovascular function by cleaving the carboxy terminal His-Leu dipeptide from angiotensin I to produce a potent vasopressor octapeptide, angiotensin II. Inhibitors of ACE are a first line of therapy for hypertension, heart failure, myocardial infarction and diabetic nephropathy. Notably, these inhibitors were developed without knowledge of the structure of human ACE, but were instead designed on the basis of an assumed mechanistic homology with carboxypeptidase A. Here we present the X-ray structure of human testicular ACE and its complex with one of the most widely used inhibitors, lisinopril (N2-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline; also known as Prinivil or Zestril), at 2.0 A resolution. Analysis of the three-dimensional structure of ACE shows that it bears little similarity to that of carboxypeptidase A, but instead resembles neurolysin and Pyrococcus furiosus carboxypeptidase--zinc metallopeptidases with no detectable sequence similarity to ACE. The structure provides an opportunity to design domain-selective ACE inhibitors that may exhibit new pharmacological profiles.

Amino Acid Sequence↗
PubMed1996

In vitro hydrogen production by glucose dehydrogenase and hydrogenase.

A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated. The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase. Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor. This simple system may provide a method for the biological production of hydrogen from renewable sources. In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity.

Cloning, Molecular↗