Rita Florio

Endocannabinoids, Related Compounds and Their Metabolic Routes

Endocannabinoids are lipid mediators able to bind to and activate cannabinoid receptors, the primary molecular targets responsible for the pharmacological effects of the Δ9-tetrahydrocannabinol. These bioactive lipids belong mainly to two classes of compounds: N-acylethanolamines and acylesters, being N-arachidonoylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), respectively, their main representatives. During the last twenty years, an ever growing number of fatty acid derivatives (endocannabinoids and endocannabinoid-like compounds) have been discovered and their activities biological is the subject of intense investigations. Here, the most recent advances, from a therapeutic point of view, on endocannabinoids, related compounds, and their metabolic routes will be reviewed.

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.

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.

The Fatty Acid Amide Hydrolase in Lymphocytes from Sedentary and Active Subjects

PURPOSE Endocannabinoids (eCB) and interleukin 6 (IL-6) levels change during physical activity, thus suggesting their involvement in the modulation of exercise-related processes like inflammation and energy homeostasis. To investigate whether lifestyle might affect the activity of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), active and sedentary subjects were enrolled. METHODS Plasma IL-6 levels and lymphocyte FAAH activity of eight physically active male subjects (mean ± SEM; age = 39.3 ± 2.9 yr, body mass index = 21.1 ± 0.4 kg·m), usually practicing aerobic exercise (8.1 ± 1.2 h·wk), and eight sedentary subjects (38.8 ± 3.7 yr, body mass index = 23.1 ± 0.8 kg·m) were measured. Also, in vitro effect of IL-6 was tested on FAAH expression and activity and on FAAH promoter activity in lymphocytes from sedentary subjects. RESULTS Under resting conditions (at least 12 h from the last exercise), the active group showed plasma IL-6 levels (2.74 ± 0.73 pg·mL) and lymphocyte FAAH activity (215.7 ± 38.5 pmol·min·mg protein) significantly higher than those measured in the sedentary group (0.20 ± 0.02 pg·mL, and 42.0 ± 4.2 pmol·min·mg protein). Increased IL-6 levels paralleled increased FAAH activity, and consistently, the in vitro treatment of lymphocytes from sedentary individuals with 10 ng·mL IL-6 for 48 h significantly increased FAAH expression and activity. Transient transfection experiments showed that IL-6 induced the expression of a reporter gene under the control of a cAMP response element-like region in the human FAAH promoter. A mutation in the same element abolished IL-6 up-regulation, demonstrating that this cytokine regulates FAAH activity at the transcriptional level. CONCLUSION IL-6 leads to activation of the FAAH promoter, thus enhancing FAAH activity that modulates the eCB tone in physically active people.

The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase

Pyridoxal 5′‐phosphate‐dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site‐directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydromethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis.

Calcium-induced tertiary structure modifications of endo-β-1,3-glucanase from Pyrococcus furiosus in 7.9 M guanidinium chloride

The family 16 endo-beta-1,3 glucanase from the extremophilic archaeon Pyrococcus furiosus is a laminarinase, which in 7.9 M GdmCl (guanidinium chloride) maintains a significant amount of tertiary structure without any change of secondary structure. The addition of calcium to the enzyme in 7.9 M GdmCl causes significant changes to the near-UV CD and fluorescence spectra, suggesting a notable increase in the tertiary structure which leads to a state comparable, but not identical, to the native state. The capability to interact with calcium in 7.9 M GdmCl with a consistent recovery of native tertiary structure is a unique property of this extremely stable endo-beta-1,3 glucanase. The effect of calcium on the thermodynamic parameters relative to the GdmCl-induced equilibrium unfolding has been analysed by CD and fluorescence spectroscopy. The interaction of calcium with the native form of the enzyme is studied by Fourier-transform infrared spectroscopy in the absorption region of carboxylate groups and by titration in the presence of a chromophoric chelator. A homology-based model of the enzyme is generated and used to predict the putative binding site(s) for calcium and the structural interactions potentially responsible for the unusual stability of this protein, in comparison with other family 16 glycoside hydrolases.

Tertiary structure in 7.9 m guanidinium chloride − the role of Glu53 and Asp287 in Pyrococcus furiosus endo-β-1,3-glucanase

The thermodynamic stability of family 16 endo‐β‐1,3‐glucanase (EC 3.2.1.39) from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild‐type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo‐β‐1,3‐glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 m guanidinium chloride. The persistence of tertiary structure in 7.9 m guanidinium chloride, a feature of the wild‐type enzyme, is observed also for the three variant proteins. The ΔGH2O values relative to the guanidinium chloride‐induced equilibrium unfolding of the three variants are approximatelty 50% lower than that of the wild‐type. The destabilizing effect of the combined mutations of the double mutant is non‐additive, with an energy of interaction of 24.2 kJ·mol−1, suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m‐values, a parameter which reflects the solvent‐exposed surface area upon unfolding. The decrease in m‐value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non‐native state in 7.9 m guanidinium chloride comparable to that induced by the presence of calcium on the wild‐type. These results suggest that the stabilization of a compact non‐native state may be a strategy for P. furiosus endo‐β‐1,3‐glucanase to thrive under adverse environmental conditions.

Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase - The role of evolutionarily conserved hydrophobic contacts

According to their fold, pyridoxal 5′‐phosphate‐dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site‐directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5′‐phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of the quaternary structure.

Crystallization and preliminary X-ray crystallographic analysis of the laminarinase endo-β-1,3-glucanase from Pyrococcus furiosus

Laminarinase endo-beta-1,3 glucanase (LamA) from Pyrococcus furiosus is an enzyme which displays its main hydrolytic activity on the 3-1,3-glucose polymer laminarin. This laminarinase is remarkably resistant to denaturation: its secondary structure is unchanged in 8 M guanidinium chloride. This protein belongs to the family 16 glycosyl hydrolases, which are enzymes that are widely distributed among bacteria, fungi and higher plants. Single crystals of P. furiosus LamAhave been obtained by the hanging-drop vapour-diffusion method using 2-methyl-2,4-pentanediol as a precipitant agent. A complete data set has been collected under cryocooling at a synchrotron source. The crystals belong to the monoclinic space group P21, with unit-cell parameters a = 44.36, b = 84.76, c = 69.23 A, a = 90, fl = 104.97, y = 90 degrees, and diffract to 2.15 A resolution.