scholarly journals The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides

2010 ◽  
Vol 76 (12) ◽  
pp. 4109-4112 ◽  
Author(s):  
Jiro Arima ◽  
Masazumi Morimoto ◽  
Hirokazu Usuki ◽  
Nobuhiro Mori ◽  
Tadashi Hatanaka
Keyword(s):  
Conversion Ratio ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Broad Specificity

ABSTRACT Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%.

scholarly journals The Biosynthetic Pathway of Major Avenanthramides in Oat

Metabolites ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 163 ◽  
Author(s):  
Zhiyong Li ◽  
Yi Chen ◽  
Dauenpen Meesapyodsuk ◽  
Xiao Qiu
Keyword(s):  
Biosynthetic Pathway ◽  
Molecular Mechanisms ◽  
Functional Characterization ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Coa Ligase ◽  
Health Promoting ◽  
Hydroxyanthranilic Acid ◽  
Coa Thioesters

Avenanthramides are a group of N-cinnamoylanthranilic acids, with health-promoting properties mainly found in oat (Avena sativa L.). However, the biosynthetic mechanism for the main three types of avenanthramides (Avn-A, Avn-B and Avn-C) is not completely understood. In the present study, we report molecular identification and functional characterization of three different types of genes from oat encoding 4-coumarate-CoA ligase (4CL), hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and a caffeoyl-CoA O-methyltransferase (CCoAOMT) enzymes, all involved in the biosynthesis of these avenanthramides. In vitro enzymatic assays using the proteins expressed in Escherichia coli showed that oat 4CL could convert p-coumaric acid, caffeic acid and ferulic acid to their CoA thioesters. Oat HHTs were only responsible for the biosynthesis of Avn-A and Avn-C using hydroxyanthranilic acid as an acyl acceptor and p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor, respectively. Avn-B was synthesized by a CCoAOMT enzyme through the methylation of Avn-C. Collectively, these results have elucidated the molecular mechanisms for the biosynthesis of three major avenanthramides in vitro and paved the way for metabolic engineering of the biosynthetic pathway in heterologous systems to produce nutraceutically important compounds and make possible genetic improvement of this nutritional trait in oat through marker-assisted breeding.

scholarly journals Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata

1990 ◽  
Vol 272 (1) ◽  
pp. 31-38 ◽  
Author(s):  
M Bafor ◽  
L Jonsson ◽  
A K Stobart ◽  
S Stymne
Keyword(s):  
Fatty Acid ◽  
Capric Acid ◽  
Decanoic Acid ◽  
Acyl Donor ◽  
Schematic Model ◽  
Acyl Acceptor ◽  
Medium Chain ◽  
Acyl Acceptors ◽  
Cuphea Lanceolata ◽  
Long Chain

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid (‘capric acid’) in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.

Relative suitability of 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glycerylphosphorylcholine and different acyl donors for phosphatidylcholine synthesis via acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes

1980 ◽  
Vol 58 (5) ◽  
pp. 434-439 ◽  
Author(s):  
B. J. Holub ◽  
J. Plekarski ◽  
F. Possmayer
Keyword(s):  
Fatty Acids ◽  
The Other ◽  
Acyl Donor ◽  
Rat Lung ◽  
Acceptor Reaction ◽  
Acyl Acceptor ◽  
Dipalmitoyl Phosphatidylcholine ◽  
Acyl Donors ◽  
Phosphatidylcholine Synthesis ◽  
Assay Conditions

The relative suitability of the 1-palmitoyl and 1-stearoyl homologues of 1-acyl-sn-glyceryl-phosphorylcholine and different acyl donors were tested as substrates for phosphatidylcholine synthesis via the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes. The acyl acceptor was an almost equi-molar mixture of the [3H]palmitoyl plus [14C]stearoyl species of 1-acyl-sn-glycero-3-phosphorylcholine with palmitoyl-, stearoyl-, oleoyl-, linoleoyl-, or arachidonoyl-CoA serving as the acyl donor. At all concentrations of acyl acceptor, reaction velocities with 20:4-CoA ≥ 18:2-CoA > 18:1-CoA > 16:0-CoA > 18:0-CoA. Furthermore, the acyltransferase selectively utilized the 1-palmitoyl over the 1-stearoyl species of 1-acylglycerylphosphorylcholine by 4.2- to 5.7-fold under optimal assay conditions with the various acyl-CoA thiolesters. However, the degree of preference exhibited for the 1-palmitoyl-sn-glycero-3-phosphorylcholine, as acyl acceptor, versus the 1-stearoyl homologue with palmitoyl-CoA as the acyl donor was not significantly different from that obtained with the other acyl-CoA derivatives. Thus, the specificity of the acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyltransferase indicates an ability of this enzyme to produce dipalmitoyl phosphatidylcholine but it cannot independently explain the predominance of dipalmitoyl phosphatidylcholine in lung or the tendency of stearate at the 1-position to associate with fatty acids of increasing unsaturation at the 2-position.

scholarly journals Versatility in acyltransferase activity completes chicoric acid biosynthesis in purple coneflower

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rao Fu ◽  
Pingyu Zhang ◽  
Ge Jin ◽  
Lianglei Wang ◽  
Shiqian Qi ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Bioactive Compound ◽  
Acyl Donor ◽  
Serine Carboxypeptidase ◽  
Acid Biosynthesis ◽  
Acyltransferase Activity ◽  
Acyl Acceptor ◽  
Chicoric Acid ◽  
Caftaric Acid ◽  
Purple Coneflower

AbstractPurple coneflower (Echinacea purpurea (L.) Moench) is a popular native North American herbal plant. Its major bioactive compound, chicoric acid, is reported to have various potential physiological functions, but little is known about its biosynthesis. Here, taking an activity-guided approach, we identify two cytosolic BAHD acyltransferases that form two intermediates, caftaric acid and chlorogenic acid. Surprisingly, a unique serine carboxypeptidase-like acyltransferase uses chlorogenic acid as its acyl donor and caftaric acid as its acyl acceptor to produce chicoric acid in vacuoles, which has evolved its acyl donor specificity from the better-known 1-O-β-D-glucose esters typical for this specific type of acyltransferase to chlorogenic acid. This unusual pathway seems unique to Echinacea species suggesting convergent evolution of chicoric acid biosynthesis. Using these identified acyltransferases, we have reconstituted chicoric acid biosynthesis in tobacco. Our results emphasize the flexibility of acyltransferases and their roles in the evolution of specialized metabolism in plants.

scholarly journals Dipeptide Synthesis by an Aminopeptidase from Streptomyces septatus TH-2 and Its Application to Synthesis of Biologically Active Peptides

2006 ◽  
Vol 72 (6) ◽  
pp. 4225-4231 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Misugi Uraji ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Methyl Ester ◽  
Free Amino ◽  
Methyl Esters ◽  
Enzyme Reaction ◽  
Biologically Active ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Active Peptides ◽  
Biologically Active Peptides ◽  
Acyl Acceptors

ABSTRACT Dipeptide synthesis by aminopeptidase from Streptomyces septatus TH-2 (SSAP) was demonstrated using free amino acid as an acyl donor and aminoacyl methyl ester as an acyl acceptor in 98% methanol (MeOH). SSAP retained its activity after more than 100 h in 98% MeOH, and in the case of phenylalanyl-phenylalanine methyl ester synthesis, the enzyme reaction reached equilibrium when more than 50% of the free phenylalanine was converted to the product. In an investigation of the specificity of SSAP toward acyl donors and acyl acceptors, SSAP showed a broad specificity toward various free amino acids and aminoacyl methyl esters. Furthermore, we applied SSAP to the synthesis of several biologically active peptides, such as aspartyl-phenylalanine, alanyl-tyrosine, and valyl-tyrosine methyl esters.

scholarly journals Microbial Transglutaminases investigations for selecting putative forms of industrial interest

2016 ◽  
Author(s):  
Deborah Giordano ◽  
Angelo Facchiano
Keyword(s):  
Clinical Applications ◽  
Amino Acid Sequences ◽  
Acyl Transfer ◽  
Acyl Donor ◽  
Post Translational Modification ◽  
Human Form ◽  
Acyl Acceptor ◽  
Functional Features ◽  
Streptomyces Mobaraensis

Motivation: Microbial Transglutaminase (MTGase) is an enzyme belonging to the class of transferases. Enzymes of this family (E.C. 2.3.2.13) catalyze post-translational modification in many proteins by acyl transfer reactions, deamidation and crosslinking (polymerisation) between protein intra- or inter-chain glutamine (acyl donor) and lysine (acyl acceptor) peptide residues. Even if the functions of transglutaminases in bacteria are unknown, now they are become an important tool for industry, but also for research and biotechnology fields. The interest in these enzymes is also focused on various biological processes and clinical applications as well as on their important role in the prevention of allergy and food intolerance, including celiac disease. Since the early 1990s, many MTGase-producing strains have been found, and production processes have been optimized. Nowadays the MTGase produced by Streptomyces mobaraensis is commercially available and widely used in biopolymers industry, in cosmetics, in clinical applications, in wool textiles, and in the food processing industry. We are interested to explore the existence of different MTGases that may maintain or improve the functional features of the mTGase currently in use. Methods: Databases searches have been performed in order to identify amino acid sequences of potential transglutaminases in microorganisms. NCBI databases, UniProt and specific microbial genome databases have been searched by querying the sequence from Streptomyces mobaraensis sequence and human tissue TGase, as reference to human form, by BLAST tools. Results: Microbial sequences annotated as “hypothetical transglutaminases” are largely present in databases. Searches based on the selected query sequences evidence a even more high number of MTGases. Among them, we find different characteristics in terms of length, presence of domains, position of amino acids with expectable functional roles. The results suggest a variety of forms within the microbial universe, and this encourage us for the search of novel proteins to be investigated for in vitro activity.

scholarly journals Remodeling of Crossbridges Controls Peptidoglycan Cross-linking Levels in Bacterial Cell Walls

2019 ◽  
Author(s):  
Sean E. Pidgeon ◽  
Alexis J. Apostolos ◽  
Marcos M. Pires
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Bacterial Species ◽  
Physiological Role ◽  
Primary Component ◽  
Acyl Donor ◽  
Bacterial Cells ◽  
Structural Variations ◽  
Acyl Acceptor ◽  
Donor And Acceptor

ABSTRACTCell walls are barriers found in almost all known bacterial cells. These structures establish a controlled interface between the external environment and vital cellular components. A primary component of cell wall is a highly crosslinked matrix called peptidoglycan (PG). PG crosslinking, carried out by transglycosylases and transpeptidases, is necessary for proper cell wall assembly. Transpeptidases, targets of β-lactam antibiotics, stitch together two neighboring PG stem peptides (acyl-donor and acyl-acceptor strands). We recently described a novel class of cellular PG probes that were processed exclusively as acyl-donor strands. Herein, we have accessed the other half of the transpeptidase reaction by developing probes that are processed exclusively as acyl-acceptor strands. The critical nature of the crossbridge on the PG peptide was demonstrated in live bacterial cells and surprising promiscuity in crossbridge primary sequence was found in various bacterial species. Additionally, acyl-acceptor probes provided insight into how chemical remodeling of the PG crossbridge (e.g., amidation) can modulate crosslinking levels, thus establishing a physiological role of PG structural variations. Together, the acyl-donor and -acceptor probes will provide a versatile platform to interrogate PG crosslinking in physiologically relevant settings.SYNOPSIS TOC

scholarly journals A Modified, Optimized Kinetic Photometric Assay for the Determination of Blood Coagulation Factor XIII Activity in Plasma

2000 ◽  
Vol 46 (12) ◽  
pp. 1946-1955 ◽  
Author(s):  
Levente Kárpáti ◽  
Botond Penke ◽  
Éva Katona ◽  
István Balogh ◽  
György Vámosi ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Factor Xiii ◽  
Clinical Chemistry ◽  
Coagulation Factor ◽  
Reference Interval ◽  
Acyl Donor ◽  
Acyl Acceptor ◽  
Coagulation Factor Xiii ◽  
Increased Sensitivity ◽  
Fxiii Activity

Abstract Background: Blood coagulation factor XIII (FXIII) is a zymogen that is transformed into an active transglutaminase by thrombin and Ca2+. FXIII plays an essential role in fibrin stabilization and in the protection of fibrin from proteolytic degradation. No convenient method has been available for the measurement of FXIII activity in plasma. The aim of the present study was to improve and optimize a kinetic photometric FXIII assay originally developed in our laboratory. Methods: In the assay, FXIII was activated by thrombin and Ca2+. Fibrin polymerization was prevented by an inhibitory tetrapeptide. Glycine-ethyl ester and a glutamine residue of a synthetic dodecapeptide served as acyl acceptor and acyl donor transglutaminase substrates, respectively. The amount of ammonia released during the reaction was monitored using glutamate dehydrogenase and NADPH. Results: The use of a new glutamine substrate and optimization of activator and substrate concentrations increased sensitivity. Substitution of NADPH for NADH and introduction of an appropriate blank eliminated systemic overestimation of FXIII activity. The recovery of FXIII was 96%, the assay was linear up to 470 U/L, the detection limit was 1 U/L, and the imprecision (CV) was <8% even at very low FXIII activities. A reference interval of 108–224 U/L (69–143%) was established. The results correlated well with results obtained by an immunoassay specific for plasma FXIII. Conclusions: The optimized FXIII assay is a simple, rapid method for the diagnosis of inherited or acquired FXIII deficiencies and increased FXIII concentrations. It can be easily adapted to clinical chemistry analyzers.

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