Valerio Consalvi

The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures

BACKGROUND The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C. The proteins from this organism display extreme thermostability. We have undertaken the structure determination of glutamate dehydrogenase from P. furiosus in order to gain further insights into the relationship between molecular structure and thermal stability. RESULTS The structure of P. furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum. CONCLUSIONS Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces. We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures.

PROTEIN THERMOSTABILITY IN EXTREMOPHILES

Thermostability of a protein is a property which cannot be attributed to the presence of a particular amino acid or to a post synthetic modification. Thermostability seems to be a property acquired by a protein through many small structural modifications obtained with the exchange of some amino acids and the modulation of the canonical forces found in all proteins such as electrostatic (hydrogen bonds and ion-pairs) and hydrophobic interactions. Proteins produced by thermo and hyperthermophilic microorganisms, growing between 45 and 110 degrees C are in general more resistant to thermal and chemical denaturation than their mesophilic counterparts. The observed structural resistance may reflect a restriction on the flexibility of these proteins, which, while allowing them to be functionally competent at elevated temperatures, renders them unusually rigid at mesophilic temperatures (10-45 degrees C). The increased rigidity at mesophilic temperatures may find a structural determinant in increased compactness. In thermophilic proteins a number of amino acids are often exchanged. These exchanges with some strategic placement of proline in beta-turns give rise to a stabilization of the protein. Mutagenesis experiments have confirmed this statement. From the comparative analysis of the X-ray structures available for several families of proteins, including at least one thermophilic structure in each case, it appears that thermal stabilization is accompanied by an increase in hydrogen bonds and salt bridges. Thermostability appears also related to a better packing within buried regions. Despite these generalisations, no universal rules can be found in these proteins to achieve thermostability.

The unusual co-assembly of H- and M-chains in the ferritin molecule from the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi

Ferritins from the liver and spleen of the cold-adapted Antarctic teleosts Trematomus bernacchii and Trematomus newnesi have been isolated and characterized. Interestingly, only H- and M-chains are expressed and no L-chains. The H-chains contain the conserved ferroxidase center residues while M-chains harbor both the ferroxidase center and the micelle nucleation site ligands. Ferritins have an organ-specific subunit composition, they are: M homopolymers in spleen and H/M heteropolymers in liver. The M-chain homopolymer mineralizes iron at higher rate with respect to the H/M heteropolymer, which however is endowed with a lower activation energy for the iron incorporation process, indicative of a higher local flexibility. These findings and available literature data on ferritin expression in fish point to the role of tissue-specific expression of different chains in modulating the iron oxidation/mineralization process.

Computational and Experimental Studies on β-Sheet Breakers Targeting Aβ1–40 Fibrils

Background: β-Amyloid aggregates are at the basis of Alzheimer disease development. Short synthetic peptides are seen to inhibit polymerization. Results: Various synthetic peptides have been studied by MD simulations and tested experimentally. Conclusion: Combined results indicate Ac-LPFFN-NH2 as an effective lead compound able to slow down Aβ1–40 aggregation. Significance: Designing potential Aβ aggregation inhibitors will help fight Alzheimer disease. In this work we present and compare the results of extensive molecular dynamics simulations of model systems comprising an Aβ1–40 peptide in water in interaction with short peptides (β-sheet breakers) mimicking the 17–21 region of the Aβ1–40 sequence. Various systems differing in the customized β-sheet breaker structure have been studied. Specifically we have considered three kinds of β-sheet breakers, namely Ac-LPFFD-NH2 and two variants thereof, one obtained by substituting the acetyl group with the sulfonic amino acid taurine (Tau-LPFFD-NH2) and a second novel one in which the aspartic acid is substituted by an asparagine (Ac-LPFFN-NH2). Thioflavin T fluorescence, circular dichroism, and mass spectrometry experiments have been performed indicating that β-sheet breakers are able to inhibit in vitro fibril formation and prevent the β sheet folding of portions of the Aβ1–40 peptide. We show that molecular dynamics simulations and far UV circular dichroism provide consistent evidence that the new Ac-LPFFN-NH2 β-sheet breaker is more effective than the other two in stabilizing the native α-helix structure of Aβ1–40. In agreement with these results thioflavin T fluorescence experiments confirm the higher efficiency in inhibiting Aβ1–40 aggregation. Furthermore, mass spectrometry data and molecular dynamics simulations consistently identified the 17–21 Aβ1–40 portion as the location of the interaction region between peptide and the Ac-LPFFN-NH2 β-sheet breaker.

Crystal structure of a family 16 endoglucanase from the hyperthermophile Pyrococcus furiosus - Structural basis of substrate recognition

Bacterial and archaeal endo‐β‐1,3‐glucanases that belong to glycoside hydrolase family 16 share a β‐jelly‐roll fold, but differ significantly in sequence and in substrate specificity. The crystal structure of the laminarinase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus (pfLamA) has been determined at 2.1 Å resolution by molecular replacement. The pfLamA structure reveals a kink of six residues (72–77) at the entrance of the catalytic cleft. This peptide is absent in the endoglucanases from alkaliphilic Nocardiopsis sp. strain F96 and Bacillus macerans, two proteins displaying an overall fold similar to that of pfLamA, but with different substrate specificity. A deletion mutant of pfLamA, lacking residues 72–75, hydrolyses the mixed‐linkage β‐1,3‐1,4‐glucan lichenan 10 times more efficiently than the wild‐type protein, indicating the importance of the kink in substrate preference.

Synthesis and activity of fibrillogenesis peptide inhibitors related to the 17–21 β-amyloid sequence

Peptide derivatives 1-5, incorporating synthetic non-proteinogenic amino acids, related to the beta-amyloid 17-21 fragment of the amyloidogenic Abeta(1-40), and the N-protected decapeptide 6, corresponding to a dimeric sequence of the same fragment, have been synthesized. These compounds were designed by using Sotos pentapeptide Ac-LPFFD-NH(2) (iAbeta5p) as lead compound. Their activity as inhibitors of fibrillogenesis and stability against enzymatic degradation have been determined. Compounds 1, 5 and 6 are potent inhibitors in comparison to the lead compound. Exposure to chymotrypsin of peptide derivatives 1-5, all containing unnatural amino acids, shows increased stability as compared with iAbeta5p and 6. Conformational properties of the new compounds have been determined by CD and FT-IR spectroscopies.

Peptidyl and azapeptidyl methylketones as substrate analog inhibitors of papain and cathepsin B

Summary Peptidyl methylketones containing Phe, Tyr, Tyr(I), Tyr(I 2 ), Leu and Ile in P 2 were synthesized and tested as substrate analog reversible inhibitors of papain and bovine spleen cathepsin B. The most effective cathepsin B inhibitor contained Tyr(I 2 ) and displayed an inhibition constant of 4.7 μM at pH 6.8 and 25°C, while Leu or Ile gave practically inert analogs. Replacement of the amino acids in P 2 with the analogous α-azaamino acids, as well as the glycine in P 1 with α-azaglycine, led to complete loss of inhibiting activity. Introducing alkoxy substituents at the methyl adjacent to the ketone group generally resulted in more effective inhibitors, with inhibition constants in the micromolar range for both papain and cathepsin B.

THE AMINO ACID SEQUENCE OF GLUTAMATE DEHYDROGENASE FROM PYROCOCCUS FURIOSUS, A HYPERTHERMOPHILIC ARCHAEBACTERIUM

The complete amino acid sequence of glutamate dehydrogenase from the archaebacteriumPyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium,Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992),Eur. J. Biochem.203, 81–87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase fromPyrococcus furiosus with that ofS. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Serine hydroxymethyltransferase from the cold adapted microorganism Psychromonas ingrahamii: a low temperature active enzyme with broad substrate specificity.

Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT) particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

DNA-binding Activity of the ERp57 C-terminal Domain Is Related to a Redox-dependent Conformational Change

ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a′ domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a′ domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a′ domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a′ domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.