Marina Porcelli

Non‐thermal effects of microwaves on proteins: thermophilic enzymes as model system

Two thermophilic and thermostable enzymes, isolated from Sulfolobus solfataricus, S‐adenosylhomocysteine hydrolase and 5′‐methylthioadenosine phosphorylase, were exposed to 10.4 GHz microwave radiation in order to discriminate between thermal and non‐thermal microwave effects. The exposure causes a non‐thermal, irreversible and time‐dependent inactivation of both enzymes; the inactivation rate is related to the energy absorbed and is independent of the enzyme concentration. The influence of salts on enzyme inactivation has also been investigated. Conformational changes of S‐adenosylhomocysteine hydrolase, detected by fluorescence and circular dichroism techniques, suggest that microwaves induce protein structural rearrangements not related to temperature.

S‐Adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus.

Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C. The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively. The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B. The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively. Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform. Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration. The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents. The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP. The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C. The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated.

Uptake of adenosylmethionine and related sulfur compounds by isolated rat liver

1. Introduction The biochemical roles of S-adenosylmethionine as donor of methyl group, propylamine moiety and aminobutyryl side chain in a variety of reactions are well established [ 1,2]. Although the biological importance of SAM is widely recognized, the mecha- nisms of transport of this highly charged molecule in mammalian cells have not yet been investigated. An active transport system with high affinity towards SAM has been described in yeast cells, which accumulate very peculiarly the sulfonium compound into the vacuoles [3,4]

Olive oil phenolic compounds inhibit homocysteine-induced endothelial cell adhesion regardless of their different antioxidant activity.

In this study, we examine the effect of extra virgin olive oil phenolic compounds on homocysteine-induced endothelial dysfunction and whether the protective effects are related to their different scavenging activities. Structurally related compounds have been assayed for their ability to reduce homocysteine-induced monocyte adhesion as well as the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in EA.hy.926 cells. As well-known, among the selected phenolic compounds, hydroxytyrosol, homovanillyl alcohol, and the hydroxycinnamic acid derivatives caffeic and ferulic acid display high scavenging activities, while tyrosol and p-coumaric acid are poorly active. All of the tested compounds, approaching potential in vivo concentrations, significantly reduce homocysteine-induced cell adhesion and ICAM-1 expression. Interestingly, we report the first evidence that monophenols tyrosol and p-coumaric acid are selectively protective only in homocysteine-activated cells, while they are ineffective in reducing ICAM-1 expression induced by TNFalpha. Finally, we report the synergistic effect of o-diphenolic and monophenolic compounds.

A novel hyperthermostable 5′-deoxy-5′-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus

We report herein the first molecular characterization of 5′‐deoxy‐5′‐methylthio‐adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 µm) for 5′‐deoxy‐5′‐methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 °C and extremely thermostable with an apparent Tm of 112 °C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride‐induced unfolding with a transition midpoint of 3.3 m after 22‐h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C‐terminal region and that the C‐terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5′‐deoxy‐5′‐methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site‐directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild‐type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5′‐deoxy‐5′‐methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5′‐deoxy‐5′‐methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.

The First Agmatine/Cadaverine Aminopropyl Transferase: Biochemical and Structural Characterization of an Enzyme Involved in Polyamine Biosynthesis in the Hyperthermophilic Archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.

S-Adenosylmethionine decarboxylase from the thermophilic archaebacterium Sulfolobus solfataricus

It is well known that S-adenosylmethionine decarboxylase (AdoMet DC) plays a key role in the polyamine biosynthetic pathway by catalyzing the formation of S-adenosyl(5′)-3-methylthiopropylamine, the donor of the propylamine moiety of polyamines (1–3).

Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus

We report here the characterization of the first mammalian‐like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5′‐deoxy‐5′‐methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6‐oxo to a 6‐amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 °C and is characterized by extreme thermodynamic stability (Tm, 110 °C that increases to 120 °C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 °C), and remarkable SDS‐resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C‐terminal region and that the C‐terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C‐terminal region may also account for the extreme enzyme thermostability.

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: Biochemical characterization and analysis of protein structure by comparative molecular modeling

S‐Adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S‐adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L‐homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95°C, and thermostable retaining 100% residual activity after 1 h at 90°C and showing an apparent melting temperature of 98°C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N‐terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C‐terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology‐modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C‐terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD‐binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures. Proteins 2005.

Biochemical characterization and homology modeling of a purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus: Insights into mechanisms of protein stabilization

We report the biochemical and structural characterization of the purine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus (SsIAG-NH). SsIAG-NH is a homodimer of 70kDa specific for adenosine, guanosine and inosine. SsIAG-NH is highly thermophilic and is characterized by extreme thermodynamic stability (T(m), 107 degrees C), kinetic stability and remarkable resistance to guanidinium chloride-induced unfolding. A disulfide bond that, on the basis of SDS-PAGE is positioned intersubunits, plays an important role in thermal stability. SsIAG-NH shares 43% sequence identity with the homologous pyrimidine-specific nucleoside hydrolase from S. solfataricus (SsCU-NH). The comparative sequence alignment of SsIAG-NH, SsCU-NH, purine non-specific nucleoside hydrolase from Crithidia fasciculata and purine-specific nucleoside hydrolase from Trypanosoma vivax shows that, only few changes in the base pocket are responsible for different substrate specificity of two S. solfataricus enzymes. The structure of SsIAG-NH predicted by homology modeling allows us to infer the role of specific residues in substrate specificity and thermostability.