Gerhard A. Venter

β-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed β-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the β-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

The mechanism of the amidases: mutating the glutamate adjacent to the catalytic triad inactivates the enzyme due to substrate mispositioning.

Background: A cysteine, a glutamic acid, and a lysine are the well known amidase catalytic residues. Results: Mutating the neighboring, structurally conserved Glu-142 inactivates the enzyme, but the active site cysteine still reacts with acrylamide via its double bond. Conclusion: Glu-142 positions the amide for productive nucleophilic attack by the cysteine. Significance: An intact catalytic tetrad is required for amidase activity. All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys “catalytic triad.” In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained despite the mutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu-59) in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 Oϵ1 in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 Oδ1. In neither case is an atom located at the position of Glu-142 Oϵ2 in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 Oϵ2 and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.

Conformational flexibility of sulphur linked saccharides a possible key to their glycosidase inhibitor activity

We investigate the reasons why sulphur linked saccharides are good inhibitors of retaining β-glycosidases. A comparison of the conformational space and electronic profile of the oxygen and sulphur linked oligosaccharides using HF/6-31G* * reveals that they are electronically very similar and have identical conformational preferences. However, the conformational barriers separating the minima are at least 3 kcal mol− 1 lower for the sulphur linkage implying a greater conformational flexibility. Furthermore, we find using natural bond orbital analysis that the sulphur linkage is significantly less open to acid hydrolysis than the oxygen, found in the natural sugar on which retaining β-glycosidases act. Our nanosecond molecular dynamics studies of Bacillus agaradhaerens glycosidase reveal that the thio-cellotriose binds the enzyme 6 kcal mol− 1more strongly than does cellotriose. A comparison of the conformational space of the sulphur linkage with that of the oxygen glycosidic linkage provides an explanation for the stronger binding which appears to be due to the greater flexibility of the sulphur glycosidic linkage.

Solution structures of chloroquine-ferriheme complexes modeled using MD simulation and investigated by EXAFS spectroscopy.

The interaction of chloroquine (CQ) and the μ-oxo dimer of iron(III) protoporphyrin IX (ferriheme) in aqueous solution was modeled using molecular dynamics (MD) simulations. Two models of the CQ-(μ-oxo ferriheme) complex were investigated, one involving CQ π-stacked with an unligated porphyrin face of μ-oxo ferriheme and the other in which CQ was docked between the two porphyrin rings. The feasibility of both models was tested by fitting computed structures to the experimental extended X-ray absorption fine structure (EXAFS) spectrum of the CQ-(μ-oxo ferriheme) complex in frozen aqueous solution. The docked model produced better agreement with experimental data, suggesting that this is the more likely structure in aqueous solution. The EXAFS fit indicated a longer than expected Fe-O bond of 1.87Å, accounting for the higher than expected magnetic moment of the complex. As a consequence, the asymmetric Fe-O-Fe stretch shifts much lower in frequency and was identified in the precipitated solid at 744cm(-1) with the aid of the O(18) isomer shift. Three important CQ-ferriheme interactions were identified in the docked structure. These were a hydrogen bond between the oxide bridge of μ-oxo ferriheme and the protonated quinolinium nitrogen atom of CQ; π-stacking between the quinoline ring of CQ and the porphyrin rings; and a close contact between the 7-chloro substituent of CQ and the porphyrin methyl hydrogen atoms. These interactions can be used to rationalize previously observed structure-activity relationships for quinoline-ferriheme association.

Molecular Structures and Solvation of Free Monomeric and Dimeric Ferriheme in Aqueous Solution: Insights from Molecular Dynamics Simulations and Extended X-ray Absorption Fine Structure Spectroscopy

CHARMM force field parameters have been developed to model nonprotein bound five-coordinate ferriheme (ferriprotoporphyrin IX) species in aqueous solution. Structures and solvation were determined from molecular dynamics (MD) simulations at 298 K of monomeric [HO-ferriheme](2-), [H2O-ferriheme](-), and [H2O-ferriheme](0); π-π dimeric [(HO-ferriheme)2](4-), [(H2O-ferriheme)(HO-ferriheme)](3-), [(H2O-ferriheme)2](2-), and [(H2O-ferriheme)2](0); and μ-oxo dimeric [μ-(ferriheme)2O](4-). Solvation of monomeric species predominated around the axial ligand, meso-hydrogen atoms of the porphyrin ring (Hmeso), and the unligated face. Existence of π-π ferriheme dimers in aqueous solution was supported by MD calculations where such dimers remained associated over the course of the simulation. Porphyrin rings were essentially coplanar. In these dimers major and minor solvation was observed around the axial ligand and Hmeso positions, respectively. In μ-oxo ferriheme, strong solvation of the unligated face and bridging oxide ligand was observed. The solution structure of the μ-oxo dimer was investigated using extended X-ray absorption fine structure (EXAFS) spectroscopy. The EXAFS spectrum obtained from frozen solution was markedly different from that recorded on dried μ-oxo ferriheme solid. Inclusion of five solvent molecules obtained from spatial distribution functions in the structure generated from MD simulation was required to produce acceptable fits to the EXAFS spectra of the dimer in solution, while the solid was suitably fitted using the crystal structure of μ-oxo ferriheme dimethyl ester which included no solvent molecules.

Investigation of sublimation with and without dissociation in the chloride and nitrate salts of 4-(1-hydroxy-1,2-diphenylethyl)pyridine

We observed sublimation–dissociation and recrystallization under ambient pressure when a single crystal of 1H+·Cl− (4-(1-hydroxy-1,2-diphenylethyl)pyridinium chloride) was heated. The native organic moiety (1) crystallized on the mother crystal on distinct surfaces, providing an excellent example of molecular structure–macroscopical property relationship which can be explained by partial isostructurality. Under similar conditions the nitrate salt of the same compound, 1H+·NO3− (4-(1-hydroxy-1,2-diphenylethyl)pyridinium nitrate), sublimed and recrystallized without dissociation. The two crystal structures of the salts are isostructural, but Hirshfeld surface analysis shows significant differences between the intermolecular interactions which explain their different thermal behavior in the crystalline phase. Computational studies in the gas phase provide a theoretical understanding of the solid state behavior.

Effect of chain length on the interactions of sodium N-alkyl prolinates with bovine serum albumin: a spectroscopic investigation and molecular docking simulations

The interaction of anionic surfactants with serum albumin has emerged as an important area of research and is considered as a model for gaining fundamental insight into surfactant-protein binding, which is useful in both chemical and biological applications. This study involves the interactions of three synthesized proline-based anionic surfactants of varying chain lengths (C8, C10, and C12) with bovine serum albumin (BSA) using different techniques, including fluorescence, 1H NMR, and FT-IR spectroscopy. The study indicated that the binding of the proline surfactants with BSA followed a static quenching process, with an increase in binding ability upon increasing chain length. FT-IR studies revealed a change in the secondary structure of BSA upon binding with the proline surfactant, while 1H NMR investigations indicated the proximity of the long alkyl chain of the surfactant with tryptophan residues of BSA. Molecular docking studies performed on the three proline surfactants with BSA revealed that the surfactants were able to bind in the vicinity of both tryptophan residues (Trp-213 and Trp-134) with an increase in the free energy of binding while increasing the chain length from C8 to C12. Therefore, this study provided a whole view about the interaction of BSA with anionic proline derived surfactants, which can be used as potential ingredients in pharmaceutical products.

Quaternized α,α′-Amino Acids via Curtius Rearrangement of Substituted Malonate–Imidazolidinones

An efficient synthesis protocol is presented for accessing quaternized α-amino acids in chiral, nonracemic form via diastereoselective malonate alkylation followed by C- to N-transposition. The key stereodifferentiating step involves a diastereoselective alkylation of an α-monosubstituted malonate-imidazolidinone, which is followed first by a chemoselective malonate PMB ester removal and then a Curtius rearrangement to provide the transposition. The method demonstrates a high product ee (89-99% for eight cases) for quaternizing a range of proteinogenic α-amino acids. The stereogenicity in targets 5a-i supports previous conclusions that the diastereoselective alkylation step proceeds via an α-substituted malonate-imidazolidinone enolate in its Z-configuration, with the auxiliary in an s-transC-N conformation.

Catalytic enantioselective acyl transfer: the case for 4-PPY with a C-3 carboxamide peptide auxiliary based on synthesis and modelling studies

A series of 4-pyrrolidinopyridine (4-PPY) C-3 carboxamides containing peptide-based side chains have been synthesised and evaluated in the kinetic resolution of a small library of chiral benzylic secondary alcohols. A key design element was the incorporation of a tryptophan residue in the peptide side chain for promoting π-stacking between peptide side chain and the pyridinium ring of the N-acyl intermediate, in which modelling was used as a structure-based guiding tool. Together, a catalyst containing a LeuTrp-N-Boc side chain (catalyst 8) was identified that achieved s-values up to and in slight excess of 10. A transition-state model based on the modelling is proposed to explain the origin of enantioselectivity. This study establishes the usefulness of modelling as a structure-based guiding tool for enantioselectivity optimization as well as the potential for developing scalable peptide-based DMAP-type catalysts for large-scale resolution work.