SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch

S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-l-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the “J1/2-J3/4” gate and “locks” down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.     

Preparation of regioselectively feruloylated p-nitrophenyl α-l-arabinofuranosides and β-d-xylopyranosides—convenient substrates for study of feruloyl esterase specificity

p-Nitrophenyl α-l-arabinofuranoside and β-d-xylopyranoside mono-O-ferulates were prepared by 4-O-acetylferuloylation of corresponding enzymatically prepared di-O-acetates followed by deacetylation. An alternative mild acylation catalysed by zinc oxide was tested on xylopyranoside derivatives. The chemoselective methanolysis of the acetyl groups using neutral catalyst dibutyltin oxide at reflux was used as deacetylation method. Under these conditions a significant feruloyl migration was observed mainly on p-nitrophenyl 3-O-feruloyl-β-d-xylopyranoside resulting in low yields of the positional isomers. Investigation of substrate and positional specificity of different types of feruloyl esterases on the presented compounds in enzyme-coupled assays was reported previously.     

Efficient synthesis of a 6-deoxytalose tetrasaccharide related to the antigenic O-polysaccharide produced by Aggregatibacter actinomycetemcomitans serotype c

Concise synthesis of a 6-deoxy-α-l-talose tetrasaccharide, 6-deoxy-α-l-Talp-(1→3)-6-deoxy-α-l-Talp-(1→2)-6-deoxy-α-l-Talp-(1→3)-6-deoxy-α-l-Talp, the dimer of the disaccharide repeating unit of the OPS from Aggregatibacter actinomycetemcomitans serotype c, has been accomplished through suitable protecting group manipulations and stereoselective glycosylation starting from commercially available l-rhamnose. The target oligosaccharide in the form of its p-methoxyphenyl glycoside is suitable for further glycoconjugate formation via selective cleavage of this group.     

Facile syntheses of acyl dihydroxyacetone phosphates and lysophosphatidic acids having different acyl groups

In this study, we report novel and simple chemical syntheses of acyl dihydroxyacetone phosphate (DHAP) and 1-acyl glycero-3-phosphate [lysophosphatidic acid (LPA)], key intermediaries in the formation of glycerolipids containing ester and ether bonds. The synthesis of acyl DHAPs involved acylating the dimethyl ketal of DHAP by acid anhydride using 4-pyrrolidinopyridine as the catalyst, and the resulting product was deketalized by HClO(4) in acetone to produce acyl DHAP. The acid anhydride was either added directly or generated in the reaction mixture from the corresponding fatty acid using dicyclohexylcarbodiimide as the condensing agent. Using these methods, a number of acyl DHAPs having short-, medium-, and long-chain saturated and unsaturated acyl groups were synthesized, with overall yields from 37% to 75%. The activities of these acyl DHAPs as substrates for guinea pig liver peroxisomal acyl DHAP:NADPH reductase and alkyl DHAP synthase were then determined. Next, starting from these acyl DHAPs, a variety of LPAs were synthesized by chemical reduction of the ketone group. Biological activities of these LPAs were determined by measuring their relative abilities to release intracellular Ca(2+) via the LPA receptor. A combined chemical-enzymatic method is also described to prepare the natural LPA from the racemic mixture.     

S‐ to N‐Acyl transfer in S‐acylcysteine isopeptides via 9‐, 10‐, 12‐, and 13‐membered cyclic transition states

S‐Acyl cysteine peptides containing α‐, β‐ or γ‐amino acid residues undergo long‐range S‐ to N‐acyl transfer to give analogs of native tripeptides and tetrapeptides containing additional carbon atoms in the chain. The ease of intramolecular S → N‐acyl transfer relative to intermolecular transacylation is favored increasingly for 9 < 12 < 13 ~ 10‐membered cyclic transition states; the observed order is explained on conformational and intermolecular interaction considerations. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Enzymatic synthesis of new aromatic and aliphatic esters of flavonoids using Candida antarctica lipase as biocatalyst

Enzymatic acylation of rutin and esculin with aromatic, aliphatic and aryl-aliphatic acids using Candida antarctica lipase in tert amyl alcohol as solvent was investigated under low water content. Whatever the acyl donor used, the conversion yields and initial rates for esculin were higher than for rutin. For a given flavonoid, the performance of these reactions depended on the acyl donor structures. For aliphatic acids, conversion yields and initial rates of both flavonoids were respectively in the ranges of 68-90% and of 9.5×10^-2-72×10^-2 mmol l^-1 h^-1. For aromatic acids, the reaction occurred only with the aryl subgroup (cinnamic, hydrocinnamic, 3,4-dihydroxyhydrocinnamic and 4-hydroxyphenyl acetic acids) and was drastically influenced by the presence of side chain and substitution patterns of the aromatic ring. Except for hydrocinnamic acid (75%, 23.4×10^-2 mmol l^-1 h^-1), with these acids the conversion yields and initial rates were lower and in the range of 10-45% and of 0.7×10^-2 to 12.1×10^-2 mmol l^-1 h^-1. Unsaturation of the side chain of the hydrocinnamic acid decreased the esculin conversion rate from 75 to 13% and initial rate from 23.4 to 1.76×10^-2 mmol l^-1 h^-1. The presence of hydroxyl or nitro-groups on the aromatic ring of the aryl aliphatic acid also reduced conversion yields and initial rates. Even without a spacer, the non-phenolic ring acid (quinic acid) was reactive and lead to conversion yields of about 20 and 23% respectively for rutin and esculin.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Chemo-enzymatic preparation of resveratrol derivatives

Regioselective derivatisation of resveratrol (1) at positions 3, 5 or 4′ was achieved by a chemo-enzymatic procedure based on standard chemical reactions and esterification or alcoholysis in organic solvents catalysed by the commercially available Pseudomonas cepacia (PcL) and Candida antarctica (CaL) lipases.     

Dipentaerythritol in the Synthesis of New-Type Phospholipids

The first representatives of phospholipids of new types were synthesized on the basis of dipentaerythritol, 2,2′-[oxybis(methylen)]bis(hydroxymethyl))-1,3-propandiol. The starting polyol was phosphorylated with hexaethylphosphoric triamide to biphosphite, which was converted into thio- and selenophosphates by treatment with sulfur or selenium. The phosphoacetals were transformed into thio- and selenoanalogues of phospholipids by direct acylation with higher fatty acid chlorides.