Diastereoselective hydrolysis of peptide esters by alkaline protease. Preparation of racemization-free peptides

A new and practical enzymatic procedure for preparative diastereoselective hydrolysis of peptide esters using the alkaline protease alcalase as a catalyst was developed. This procedure has been successfully applied to the resolution of peptide diastereomers and synthesis of racemization free peptides.     

Novel regulatory interactions and activities of mammalian tRNA synthetases

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to their cognate tRNAs, thereby ensuring the faithful translation of genetic code. In addition to their enzymatic function, these enzymes have been discovered to regulate various cellular functions such as tRNA export, ribosomal RNA synthesis, apoptosis, inflammation and angiogenesis in mammalian. The insights into the noncanonical activities of these enzymes have been obtained from their unique cellular localization, interacting partners, isoform generation and expression control. Mammalian ARSs also form a macromolecular protein complex with a few auxiliary factors. Although the physiological significance of this complex is poorly understood, it also supports the potential of mammalian ARSs as sophisticated multifunctional proteins for regulating various cellular procedures. In this review, the novel regulatory activities of mammalian ARSs will be discussed in different biological processes.     

The interaction of wheat germ tyrosyl-tRNA synthetase and the tRNA-like end of brome mosaic virus RNA has no effect on in vitro viral protein synthesis and on in vitro encapsidation.

The effect of aminoacylation of the tRNA-like end of brome mosaic virus RNA during in vitro protein synthesis and in vitro viral encapsidation was investigated. The components of the homologous system were: BMV RNA, wheat germ cell-free protein synthesizing system and pure tyrosyl-tRNA synthetase from wheat germ. During in vitro protein synthesis directed with tyrosylated as well as non-tyrosylated BMV RNA, no differences were observed in the amount and in the class of polypeptides formed neither in the velocity of the translation reaction. Excess active TyrRS was added during in vitro translation, without modifying the translation efficiency. BMV RNA and active TyrRS were preincubated prior to translation in order to interact without the translation system components and then subjected to translation in vitro. Similar results were obtained when BMV RNA was preincubated with inactive TyrRS or BSA. These results indicate that the aminoacylation of BMV RNA has no pronounced effect on viral protein synthesis in vitro. During BMV RNA encapsidation either tyrosylated or non-tyrosylated BMV RNA 4 could be encapsidated in a similar way.     

An enzymatic glycosylation of nucleoside analogues using β-galactosidase from Escherichia coli

A new enzymatic method for the synthesis of β-galactosides of nucleosides and acyclic nucleoside analogues has been developed, using β-galactosidase from Escherichia coli as a catalyst and lactose as a sugar donor. The method is very rapid, feasible and last but not least inexpensive. Its applicability has been proven for a broad variety of possible substrates with respect to its scaling up for preparative use. Five new compounds from a series of nucleoside and acyclic nucleoside analogues have been prepared on a scale of several hundred milligrams, in all cases revealing very good results of the method concerning the reproducibility of the reaction yields and simplicity of the purification process.     

Multi-enzymatic glucosylation using Eucalyptus UDP-glucosyltransferase coupled UDPglucose-fermentation by bakers' yeast.

The enzymatic synthesis of glucoside compounds using a membrane-associated UDP-glucosyltransferase fraction from Eucalyptus perriniana cultured cells as a water-insoluble catalyst (N. Nakajima, et. al., J. Ferment. Bioeng., 84 (5), pp. 455-460, 1997) has been effectively done by coupling UDPglucose-fermentation by bakers' yeast. For example, beta-thujaplicin (hinokitiol) and p-aminobenzoic acid were converted respectively to their corresponding beta-D-monoglucosides with the conversion rate of around 24-26% by the multi-enzymatic system with UDPglucose as a glucose donor, which is produced by yeast cells from glucose and 5'-UMP. Addition of either cellobiose, a substrate of beta-glucosidase, or DL-1,2-anhydro-myo-inositol, an inhibitor for the enzyme in the reaction mixture, could increased the yield of these beta-D-monoglucosides. This new enzymatic system could also be used for the synthesis of flavonoid glucosides such as isoquercitrin (quercetin 3-O-beta-D-glucoside).     

Interaction network of human aminoacyl-tRNA synthetases and subunits of elongation factor 1 complex

Aminoacyl-tRNA synthetases (ARSs) ligate amino acids to their cognate tRNAs. It has been suggested that mammalian ARSs are linked to the EF-1 complex for efficient channeling of aminoacyl tRNAs to ribosome. Here we systemically investigated possible interactions between human ARSs and the subunits of EF-1 (alpha, beta, gamma, and delta) using a yeast two-hybrid assay. Among the 80 tested pairs, leucyl- and histidyl-tRNA synthetases were found to make strong and specific interaction with the EF-1gamma and beta while glu-proly-, glutaminyl-, alanyl-, aspartyl-, lysyl-, phenylalanyl-, glycyl-, and tryptophanyl-tRNA synthetases showed moderate interactions with the different EF-1 subunits. The interactions of leucyl- and histidyl-tRNA synthetase with the EF-1 complex were confirmed by immunoprecipitation and in vitro pull-down experiments. Interestingly, the aminoacylation activities of these two enzymes, but not other ARSs, were stimulated by the cofactor of EF-1, GTP. These data suggest that a systematic interaction network may exist between mammalian ARSs and EF-1 subunits probably to enhance the efficiency of in vivo protein synthesis.     

A New Enzymatic Reaction: Enzyme Catalyzed Ammonolysis of Carboxylic Esters

A new enzymatic reaction of carboxylic esters and ammonia (ammonolysis) was studied. This reaction provides a synthetically useful and mild alternative for the synthesis of amides. Several lipases and one esterase acted as catalyst. Ammonolysis of esters of chiral carboxylic acids gave higher ee values than hydrolysis under comparable reaction conditions. Furthermore, consecutive enzymatic esterification and ammonolysis provided a convenient one-pot synthesis of carboxylic amides from carboxylic acids.     

Kinetically controlled synthesis of dipeptides using ficin as biocatalyst.

The application of the sulfhydryl protease ficin as biocatalyst is proposed as a novel method for enzyme-catalyzed synthesis of dipeptides. The negligible peptidase but considerable esterase activity at alkaline pH facilitated the kinetically controlled formation of peptide bonds by coupling the ester substrates Z-Ala-OMe and Z-Gly-OMe with L-alanine, D-alanine, L-glutamine, D-glutamine and L-Cys(acetamidomethyl) respectively. The reaction is accomplished without the occurrence of secondary peptide hydrolysis. Under optimum reaction conditions (pH 9.2, high ratio nucleophile/carboxyl component, 4.8% ethanol, 40 degrees C), the peptide yields ranged from 5 to 91%, depending on the structure of the amino and/or carboxyl component. No racemization was observed in the enzymatic reaction. Application of short-chain peptides has been advocated recently in clinical nutrition. Ficin-catalyzed peptide synthesis might be an attractive biotechnological approach for the synthesis of suitable dipeptides in this respect.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

One-pot synthesis of 3,4-dihydropyrimidin-2(1H)-ones using CuBr2 as catalyst

A simple, efficient procedure and improved conditions have been found to carry out the Biginelli reaction for the synthesis of 3,4-dihydropyrimidin-2(1H)-one derivatives. This synthesis was performed using CuBr2 as the catalyst in ethanol solution. The optimum conditions were as follows: the molar ratio of aldehyde to alpha,beta-diketone to urea or thiourea is 1:1:1:0.5, the molar ratio of catalyst to aldehyde is 25%, and the reaction time is 4 h.Under the above conditions, the highest yield of dihydropyrimidinones was up to 95%. Compared with the classical Biginelli reaction conditions, this new method has the advantage of excellent yields and short reaction times.     

Formation of peptide bonds by carboxypeptidase c from orange leaves.

Carboxypeptidase c partially purified from orange leaves was studied as a catalyst for enzymatic peptide synthesis. Various N-protected ester- and nucleophile compounds were evaluated in order to determine the substrate specificity. For further characterization of the synthetic reaction, optimum pH and the influence of the N-terminal protecting group were studied. Kinetic investigations revealed considerable differences in Km and Vmax for the nucleophile when the N-terminal protecting group of the substrate was varied.     

Computational modeling and molecular dynamics simulations of mammalian cytoplasmic tyrosyl-tRNA synthetase and its complexes with substrates

Cytoplasmic tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. TyrRSs of pathogenic organisms have gained attention as potential targets for drug development. Identifying structural differences between various TyrRSs will facilitate the development of specific inhibitors for the TyrRSs of pathogenic organisms. However, there is a deficiency in structural data for mammalian cytoplasmic TyrRS in complexes with substrates. In this work, we constructed spatial structure of full-length Bos taurus TyrRS (BtTyrRS) and its complexes with substrates using the set of computational modeling techniques. Special attention was paid to BtTyrRS complexes with substrates [L-tyrosine, K⁺ and ATP:Mg²⁺] and intermediate products [tyrosyl-adenylate (Tyr-AMP), K⁺ and PP_i:Mg²⁺] with the different catalytic loop conformations. In order to analyze their dynamical properties, we performed 100 ns of molecular dynamics (MD) simulations. MD simulations revealed new structural data concerning the tyrosine activation reaction in mammalian TyrRS. Formation of strong interaction between Lys154 and γ-phosphate suggests the additional role of CP1 insertion as an important factor for ATP binding. The presence of a potassium-binding pocket within the active site of mammalian TyrRS compensates the absence of the second lysine in the KMSKS motif. Our data provide new details concerning a role of K⁺ ions at different stages of the first step of the tyrosylation reaction, including the coordination of substrates and involvement in the PP_i releasing. The results of this work suggest that differences between ATP-binding sites of mammalian and bacterial TyrRSs are meaningful and could be exploited in the drug design.     

Structure-based mutagenesis approaches toward expanding the substrate specificity of D-2-deoxyribose-5-phosphate aldolase

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.     

海藻酸钠-壳聚糖固定化木瓜蛋白酶催化内吗啡肽的合成

反应体系以乙腈作为有机介质,在微水有机溶剂体系中以Boc-Trp-OH和Phe-NH2为底物,用海藻酸钠 壳聚糖固定化木瓜蛋白酶催化合成Trp-Phe-NH2时,产率为27.8%．在这一合成反应中,对pH值、离子强度、溶液含量、反应温度、酶用量和反应时间进行正交试验,证明pH是本合成过程的最重要影响因素．反应体系以乙腈为有机介质,在微水有机溶剂体系中以 Boc-Tyr-Pro-OMe和Trp-Phe-NH2为底物,用IPSAC催化合成Tyr-Pro-Trp-Phe-NH2,产率为35~2%．     

Reaction Medium Selection for An Enzymatic Peptide Synthesis in An Aqueous-Organic Two-Phase System

Efficient development of enzymatic synthesis in two-phase systems is closely related with appropriate selection of the reaction medium (especially the solvent and phase ratio). A selection procedure based on the calculation of the theoretically allowable conversion and product concentration is presented and applied to a peptide synthesis using papain. For the synthesis of the dipeptide Boc-Gly-Phe-OMe, the operating conditions have been determined, and the two-phase system to be used has been successfully selected (with trichloroethylene being the best solvent).     

Integrated process for the enzymatic synthesis of the octapeptide PhAcCCK-8

A process for the enzymatic synthesis of PhAcCCK-8 is presented. The CCK-8 (CCK(26-33)) peptide fragment is the minimum sequence with biological activity of the cholecystokinin hormone. A synthetic convergent strategy has been developed starting from amino acid derivatives as raw materials, employing proteases as biocatalysts for each peptide coupling. The enzymes have been immobilized by deposition onto solid supports in order to be employed in organic media at low water activity. N-terminal protecting groups such as PhAc, which can be introduced and removed enzymatically, have been employed. The synthesis process has been set up at preparative level with focus in the integration of reaction and separation steps with an overall yield of 15%.     

Automated solid-phase synthesis of metabolically stabilized triazolo-peptidomimetics

The use of 1,4-disubstituted 1,2,3-triazoles as trans-amide bond surrogates has become an important tool for the synthesis of metabolically stabilized peptidomimetics. These heterocyclic bioisosters are generally incorporated into the peptide backbone by applying a diazo-transfer reaction followed by CuAAC (click chemistry) with an α-amino alkyne. Even though the manual synthesis of backbone-modified triazolo-peptidomimetics has been reported by us and others, no procedure has yet been described for an automated synthesis using peptide synthesizers. In order to efficiently adapt these reactions to an automated setup, different conditions were explored, putting special emphasis on the required long-term stability of both the diazo-transfer reagent and the Cu(I) catalyst in solution. ISA·HCl is the reagent of choice to accomplish the diazo-transfer reaction; however, it was found instable in DMF, the most commonly used solvent for SPPS. Thus, an aqueous solution of ISA·HCl was used to prevent its degradation over time, and the composition in the final diazo-transfer reaction was adjusted to preserve suitable swelling conditions of the resins applied. The CuAAC reaction was performed without difficulties using [Cu (CH₃CN)₄]PF₆ as a catalyst and TBTA as a stabilizer to prevent oxidation to Cu(II). The optimized automated two-step procedure was applied to the synthesis of structurally diverse triazolo-peptidomimetics to demonstrate the versatility of the developed methodology. Under the optimized conditions, five triazolo-peptidomimetics (8–5 amino acid residues) were synthesized efficiently using two different resins. Analysis of the crude products by HPLC-MS revealed moderate to good purities of the desired triazolo-peptidomimetics (70–85%). The synthesis time ranged between 9 and 12.5 h.     

Enzymatic attachment of glycosaminoglycan chain to peptide using the sugar chain transfer reaction with endo-beta-xylosidase.

Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.     

The Aminoacyl-tRNA Synthetase and tRNA Expression Levels Are Deregulated in Cancer and Correlate Independently with Patient Survival

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes that load amino acids to their cognate tRNA molecules. The expression of certain ARSs and tRNAs has been shown to be deregulated in cancer, presumably to accommodate elevated protein synthesis requirements. In this work, the expression of cytoplasmic ARSs and tRNAs in ten TCGA cancers has been systematically examined. ARSs were found to be mostly upregulated in tumours and their upregulation often correlated with worse patient survival. tRNAs were found to be either upregulated or downregulated in tumours and their expression sometimes correlated to worse survival outcomes. However, although the expression of most ARSs and tRNAs was deregulated in tumours when compared to healthy adjacent tissues, only in a few cases, and independently, did it correlate to patient survival. These data point to the general uncoupling of concomitant ARS and tRNA expression deregulation and patient survival, highlighting the different ARS and tRNA requirements in cancers.     

Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation

Aminoacyl tRNA synthetases (ARS) catalyze the ligation of amino acids to cognate tRNAs. Chordate ARSs have evolved distinctive features absent from ancestral forms, including compartmentalization in a multisynthetase complex (MSC), noncatalytic peptide appendages, and ancillary functions unrelated to aminoacylation. Here, we show that glutamyl-prolyl-tRNA synthetase (GluProRS), a bifunctional ARS of the MSC, has a regulated, noncanonical activity that blocks synthesis of a specific protein. GluProRS was identified as a component of the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex by RNA affinity chromatography using the ceruloplasmin (Cp) GAIT element as ligand. In response to IFN-gamma, GluProRS is phosphorylated and released from the MSC, binds the Cp 3'-untranslated region in an mRNP containing three additional proteins, and silences Cp mRNA translation. Thus, GluProRS has divergent functions in protein synthesis: in the MSC, its aminoacylation activity supports global translation, but translocation of GluProRS to an inflammation-responsive mRNP causes gene-specific translational silencing.