Regioselective monoacylation of cyclomaltoheptaose at the C-2 secondary hydroxyl groups by the alkaline protease from Bacillus subtilis in nonaqueous media

Transesterification of cyclomaltoheptaose (beta-CD) with divinyl butanedioate, divinyl hexanedioate, and divinyl decanedioate, catalyzed by the alkaline protease from Bacillus subtilis in anhydrous DMF for 5 days, furnished the corresponding vinyl-beta-CD derivatives. The products were characterized by ESI-MS, (1)H NMR, (13)C NMR, IR, and DSC. The results indicated the products to be monosubstituted esters, with monoacylation occurring at the C-2 secondary hydroxyl groups of beta-CD. The regioselectivity of the monoacylation as catalyzed by alkaline protease was not affected by the chain length of the acyl donor.     

Controlling enzyme-catalyzed regioselectivity in sugar ester synthesis

The rational control over enzyme-catalyzed regioselectivity has been studied using sucrose acylation by vinyl esters in organic media as a model. Subtilisins BPN' and Carlsberg preferentially acylate at the 1'-hydroxyl of sucrose with some acylation observed at the 6-hydroxyl. The preference for the 1'-hydroxyl is strongly affected by the hydrophobicity of the organic solvent and the chain length of the vinyl ester. Increasingly hydrophobic solvents and longer chain lengths lower the favorable formation of the 1'-acylation and improve 6-acylation. Molecular modeling of sucrose in the binding pocket of subtilisin BPN' shows that the 1'-acylation is favored in solvents that can solvate sugars (such as pyridine) as the glucose moiety is exposed to the medium, whereas 6-acylation leaves the entire sucrose molecule buried within the enzyme's binding pocket. Thus, 1'-acylation is sterically more favorable than 6-acylation. Increasingly hydrophobic solvents affect regioselectivity by changing the degree of solvation of the glucose moiety in the medium and forcing the sucrose 1'-ester completely into the binding pocket. In a related modeling, the vinyl ester chain length was shown to modulate regioselectivity by controlling the bond angles between the resulting acylenzymes and the sucrose thereby affecting the positioning of the sucrose in the binding pocket of subtilisin BPN'. This study shows that control over enzymic regioselectivity can be achieved by rational choices of substrate and solvent. (c) 1995 John Wiley & Sons, Inc.     

Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C₁₄-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C₁₄-CoA and can also bind the far more abundant palmitoyl-CoA (C₁₆-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C_18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C₁₆-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C₁₆-CoA. These results indicate that ACBD6 can locally sequester C₁₆-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C₁₆-CoA.     

Identification of dually acylated proteins from complementary DNA resources by cell-free and cellular metabolic labeling

To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP were generated and their susceptibility to protein N-myristoylation and S-palmitoylation were evaluated by metabolic labeling with [³H]myristic acid or [³H]palmitic acid either in an insect cell-free protein synthesis system or in transfected mammalian cells. As a result, EEPD1, one of five proteins (RFTN1, EEPD1, GNAI1, PDE2A, RNF11) found to be dually acylated, was shown to be a novel dually fatty acylated protein. Metabolic labeling experiments using G2A and C7S mutants of EEPD1-EGFP revealed that the palmitoylation site of EEPD1 is Cys at position 7. Analysis of the intracellular localization of EEPD1 C-terminally tagged with FLAG tag or EGFP and its G2A and C7S mutants revealed that the dual acylation directs EEPD1 to localize to the plasma membrane. Thus, dually fatty acylated proteins can be identified from cDNA resources by cell-free and cellular metabolic labeling of N-myristoylated proteins with Cys residue(s) close to the N-myristoylated N-terminus.     

The Acyl-Proteome of Syntrophus aciditrophicus Reveals Metabolic Relationships in Benzoate Degradation

Syntrophus aciditrophicus is a model syntrophic bacterium that degrades fatty and aromatic acids into acetate, CO₂, formate, and H₂ that are utilized by methanogens and other hydrogen-consuming microbes. S. aciditrophicus benzoate degradation proceeds by a multistep pathway with many intermediate reactive acyl-coenzyme A species (RACS) that can potentially N^ε-acylate lysine residues. Herein, we describe the identification and characterization of acyl-lysine modifications that correspond to RACS in the benzoate degradation pathway. The amounts of modified peptides are sufficient to analyze the post-translational modifications without antibody enrichment, enabling a range of acylations located, presumably, on the most extensively acylated proteins throughout the proteome to be studied. Seven types of acyl modifications were identified, six of which correspond directly to RACS that are intermediates in the benzoate degradation pathway including 3-hydroxypimeloylation, a modification first identified in this system. Indeed, benzoate-degrading enzymes are heavily represented among the acylated proteins. A total of 125 sites were identified in 60 proteins. Functional deacylase enzymes are present in the proteome, indicating a potential regulatory system/mechanism by which S. aciditrophicus modulates acylation. Uniquely, N^ε-acyl-lysine RACS are highly abundant in these syntrophic bacteria, raising the compelling possibility that post-translational modifications modulate benzoate degradation in this and potentially other, syntrophic bacteria. Our results outline candidates for further study of how acylations impact syntrophic consortia.     

Enantioselective resolution of 2-(1-hydroxy-3-butenyl)-5-methylfuran by immobilized lipase

An efficient and convenient strategy for synthesis of enantiomerically pure S-2-(1-hydroxy-3-butenyl)-5-methylfuran was for the first time described utilizing a lipase-mediated asymmetric acylation in organic solvents. Rhizopus arrhizus lipase was chosen as the biocatalyst, and different immobilization methods including sol–gel encapsulation and covalent attachment were adopted to improve its catalytic characteristics. Various influential factors of the reaction were also investigated. Finally, the results showed that the lipase covalently attached onto epoxy resin exhibited the highest enantioselectivity and operational stability. Under optimized reaction conditions, i.e., n-hexane as the solvent, 5/1 (mol/mol) of vinyl acetate to 2-(1-hydroxy-3-butenyl)-5-methylfuran and 30 °C, the ee value of S-1 reached up to above 98% at 52% conversion with an E value of 99.     

Solvent-free enzymatic synthesis of feruloylated diacylglycerols and kinetic study

The enzymatic esterification of glyceryl ferulate (FG) and oleic acid (OA) for feruloylated diacylglycerols (FDAG) synthesis in a solvent-free system was studied in this work. The reactions were catalyzed by different commercially available lipases, among which Novozym 435 was found to be the most active biocatalyst. The effects of glycerol in the reaction mixture and various synthesis parameters on yield of FDAG and the initial reaction rate were studied. The optimum synthesis conditions were as follows: temperature, 65 °C; enzyme load, 7.5%; substrate ratio, 7.5:1 (OA/(FG + glycerol), w/w); and reaction time, 12 h. Under the optimum conditions, the conversion of FG and yield of FDAG reached 98.0 ± 1.0% and 82.6 ± 2.2%, respectively. A linear relationship was established between the initial reaction rate and enzyme load up to 10%, which demonstrated that the influence of external mass transfer limitations on the reaction could be eliminated. The relationship between initial reaction rate and temperature was also established, based on the Arrhenius law. Novozym 435 in the present work can be used 18 times under the optimum conditions without essential losses in activity. The reaction kinetics agrees with the Ping-Pong Bi-Bi mechanism characterized by V_m and K^′_m values of 5.26 × 10⁻⁴ mol/(L min) and 0.26 mol/L, respectively.     

Multi-domain Packing in the Aminoacylatable 3′ End of a Plant Viral RNA

Turnip yellow mosaic virus (TYMV) contains a tRNA-like structure (TLS) in its 3′ untranslated region (3′ UTR).  This highly structured element induces valylation of the viral RNA by host cell enzymes and is important for virus proliferation. Directly upstream of the TYMV TLS is an upstream pseudoknot domain (UPD) that has been considered to be structurally distinct from the TLS.  However, using a combination of functional, biochemical, and biophysical assays, we show that the entire 3′ UTR of the viral genome is a single structured element in the absence of cellular protein.  This packing architecture stabilizes the RNA structure and creates a better substrate for aminoacylation, and thus the UPD and TLS are functionally and structurally coupled.  It has been proposed that the TYMV TLS acts as a molecular switch between translation and replication. Our results suggest that this putative switch could be based on structural changes within the global architecture of the UTR induced by interactions with the ribosome. The TYMV TLS·UPD might demonstrate how RNA structural plasticity can play a role in regulation of biological processes.     

Syntheses and activities of backbone‐side chain cyclic octapeptide ligands with N‐functionalized phosphotyrosine for the N‐terminal SH2‐domain of the protein tyrosine phosphatase SHP‐1

The protein tyrosine phosphatase SHP‐1 plays an important role in many physiological and pathophysiological processes. This phosphatase is activated through binding of ligands to its SH2‐domains, mainly to the N‐terminal one. Based on a theoretical docking model, backbone‐to‐side chain cyclized octapeptides were designed as ligands. Assembly of such modelled structures required the synthesis of N‐functionalized tyrosine derivatives and their incorporation into the sequence. Because of difficulties encountered in the condensation of N‐protected amino acids to the N‐alkylated tyrosine‐peptide we synthesized and used preformed dipeptide building units. As all attempts to obtain phosphorylated dipeptide units failed, the syntheses had to be performed with a free phenolic function. Use of different N‐alkyl or cycloalkyl residues in the N‐functionalized side chains allowed to investigate the effect of ring size, flexibility and hydrophobicity of formed lactam bridges on stimulatory activity. All tested linear and cyclic octapeptides stimulate the phosphatase activity of SHP‐1. Stimulatory activities of cyclic ligands increase with the chain length of the lactam bridges resulting in increased flexibility and better entropic preformation of the binding conformation. The strong activity of some cyclic octapeptides supports the modelled structure. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Efficient kinetic resolution of (R,S)-1-trimethylsilylethanol via lipase-mediated enantioselective acylation in ionic liquids

Efficient enantioselective acylation of (R,S)-1-trimethylsilylethanol {(R,S)-1-TMSE} with vinyl acetate catalyzed by immobilized lipase from Candida antarctica B (i.e., Novozym 435) was successfully conducted in ionic liquids (ILs). A remarkable enhancement in the initial rate and the enantioselectivity of the acylation was observed by using ILs as the reaction media when compared to the organic solvents tested. Also, the activity, enantioselectivity, and thermostability of Novozym 435 increased with increasing hydrophobicity of ILs. Of the six ILs examined, the IL C4MIm.PF6 gave the fastest initial rate and the highest enantioselectivity, and was consequently chosen as the favorable medium for the reaction. The optimal molar ratio of vinyl acetate to (R,S)-1-TMSE, water activity, and reaction temperature range were 4:1, 0.75, and 40 -50 degrees C, respectively, under which the initial rate and the enantioselectivity (E value) were 27.6 mM/h and 149, respectively. After a reaction time of 6 h, the ee of the remaining (S)-1-TMSE reached 97.1% at the substrate conversion of 50.7%. Additionally, Novozym 435 was effectively recycled and reused in C4MIm.PF6 for five consecutive runs without substantial lose in activity and enantioselectivity. The preparative scale kinetic resolution of (R,S)-1-TMSE in C4MIm.PF6 is shown to be very promising and useful for the industrial production of enantiopure (S)-1-TMSE.