A Substrate-induced Switch in the Reaction Mechanism of a Thermophilic Esterase KINETIC EVIDENCES AND STRUCTURAL BASIS

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites

A Late-Stage Synthetic Approach to Lanthionine-Containing Peptides via S-Alkylation on Cyclic Sulfamidates Promoted by Molecular Sieves

A one-pot, high-yield procedure for synthesizing lanthionine-containing peptides was developed. It relies on the S-alkylation of cysteine-containing peptides with chiral cyclic sulfamidates. The key feature of this approach is the use of mild reaction conditions (only activated molecular sieves are employed as the catalyst), leading to good chemoselectivity and excellent stereochemical control. The potential of the new methodology has been investigated by synthesizing the thioether ring of a natural lantibiotic, Haloduracin \u3b2

Crystallization and preliminary X-ray diffraction studies of a D

A 10-mer duplex formed between a PNA containing a `chiral box' of three adjacent d-Lys-based monomers and its complementary DNA strand has been crystallized for the first time. Crystals have been obtained using PEG 8000 as precipitant and cacodylate at pH 6.3 as buffer. The crystals belong to the space group P31 or to its enantiomorph P32, with unit-cell parameters a = b = 35.00, c = 35.91 Å. A complete data set has been collected at the synchrotron source Elettra in Trieste to 1.85 Å resolution, using a single frozen crystal

Molecular Resonance Imaging of the CAIX Expression in Mouse Mammary Adenocarcinoma Cells

The carbonic anhydrase isoform IX (hCAIX) is one of the main players in extracellular tumor pH regulation, and it is known to be overexpressed in breast cancer and other common tumors. hCA IX supports the growth and survival of tumor cells, and its expression is correlated with metastasis and resistance to therapies, making it an interesting biomarker for diagnosis and therapy. The aim of this work deals with the development of an MRI imaging probe able to target the extracellular non-catalytic proteoglycan-like (PG) domain of CAIX. For this purpose, a specific nanoprobe, LIP_PepC, was designed by conjugating a peptidic interactor of the PG domain on the surface of a liposome loaded with Gd-bearing contrast agents. A Mouse Mammary Adenocarcinoma Cell Line (TS/A) was chosen as an in vitro breast cancer model to test the developed probe. MRI results showed a high selectivity and sensitivity of the imaging probe toward hCAI-expressing TS/A cells. This approach appears highly promising for the in vivo translation of a diagnostic procedure based on the targeting of hCA IX enzyme expression

Chemoselective Glycosylation of Peptides via S\u2010Alkylation Reaction

An efficient and fast procedure in execution for synthesizing S\u2010linked glycopeptides is reported. It uses activated molecular sieves as a base to promote the selective S\u2010alkylation of readily prepared cysteine\u2010containing peptides, upon reaction of appropriate glycosyl halides. Due to the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in a satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar\u2010peptide conjugates obtained from \u3b1\u2010D\u2010glycosyl derivatives adopt a beta\u2010S\u2010conformation, indicating the high stereoselectivity of the substitution reaction

Glucan Particles Loaded with Fluorinated Emulsions: A sensitivity Improvement for the visualization of Phagocytic Cells by 19F-MRI.

A protocol to load perfluoro-crown-ether (PFCE) nanoemulsion directly into yeast-derived glucan particles (GPs) was developed. It was observed that the PFCE encapsulation did not affect the 19F-MRI properties of the nanoemulsion that is currently in clinical trials. GPs loaded with PFCE nanoemulsion were taken up avidly by murine macrophages in vitro, resulting in a cellular uptake 150 % higher than the not GPs-entrapped nanoemulsion. Accordingly, a corresponding improvement in the 19F-MRI detection of the labelled cells can be obtained. The high biotolerability and versatility of GPs, make these microcarriers a promising option for designing of improved in vivo cellular imaging protocols

Crystallization and preliminary X-ray diffraction studies of a D-lysine-based chiral PNA–DNA duplex

A 10-mer duplex formed between a PNA containing a `chiral box' of three adjacent d-Lys-based monomers and its complementary DNA strand has been crystallized for the first time. Crystals have been obtained using PEG 8000 as precipitant and cacodylate at pH 6.3 as buffer. The crystals belong to the space group P31 or to its enantiomorph P32, with unit-cell parameters a = b = 35.00, c = 35.91 Å. A complete data set has been collected at the synchrotron source Elettra in Trieste to 1.85 Å resolution, using a single frozen crystal