Threonine Aldolase Research Articles

Chemoenzymatic synthesis of 3-ethyl-2,5-dimethylpyrazine by L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate CoA ligase/L-threonine aldolase

Pyrazines are typically formed from amino acids and sugars in chemical reactions such as the Maillard reaction. In this study, we demonstrate that 3-ethyl-2,5-dimethylpyrazine can be produced from L-Thr by a simple bacterial operon. We conclude that EDMP is synthesized chemoenzymatically from L-Thr via the condensation reaction of two molecules of aminoacetone and one molecule of acetaldehyde. Aminoacetone is supplied by L-threonine 3-dehydrogenase using L-Thr as a substrate via 2-amino-3-ketobutyrate. Acetaldehyde is supplied by 2-amino-3-ketobutyrate CoA ligase bearing threonine aldolase activity from L-Thr when CoA was at low concentrations. Considering the rate of EDMP production, the reaction intermediate is stable for a certain time, and moderate reaction temperature is important for the synthesis of EDMP. When the precursor was supplied from L-Thr by these enzymes, the yield of EDMP was increased up to 20.2%. Furthermore, we demonstrate that this reaction is useful for synthesizing various alkylpyrazines.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Sustainable one-pot chemo-enzymatic synthesis of chiral furan amino acid from biomass via magnetic solid acid and threonine aldolase

Sustainable synthesis of valuable noncanonical amino acids from renewable feedstocks is of great importance. Here, a feasible chemo-enzymatic procedure was developed for the synthesis of chiral β-(2-furyl)serine from biomass catalyzed by a solid acid catalyst and immobilized E. coli whole-cell harboring l-threonine aldolase. A novel magnetic solid acid catalyst Fe3O4@MCM-41/SO42- was successfully synthesized for conversion of corncob into furfural in an aqueous system. Under the optimum conditions, furfural yield of 63.6% was achieved in 40 min at 180 ℃ with 2.0% catalyst (w/w). Furthermore, biomass-derived furfural was converted into an aldol-addition product β-(2-furyl)serine with 73.6% yield, 99% ee and 20% de by immobilized cells in 6 h. The magnetic solid acid and biocatalyst can be readily recovered and efficiently reused for five consecutive cycles without significant loss on product yields. This chemo-enzymatic route can be attractive for producing noncanonical amino acids from biomass.

Cβ-Selective Aldol Addition of d-Threonine Aldolase by Spatial Constraint of Aldehyde Binding

Cβ-Selective Aldol Addition of <scp>d</scp>-Threonine Aldolase by Spatial Constraint of Aldehyde Binding

Improving the Cβ Stereoselectivity of l-Threonine Aldolase for the Synthesis of l-threo-4-Methylsulfonylphenylserine by Modulating the Substrate-Binding Pocket To Control the Orientation of the Substrate Entrance.

l-Threonine aldolase from Actinocorallia herbida (AhLTA) is an ideal catalyst for producing l-threo-4-methylsulfonylphenylserine [(2S,3R)-1 b], a key chiral precursor for florfenicol and thiamphenicol. The moderate Cβ stereoselectivity is the main obstacle to the industrial application of AhLTA. To address this issue, a combinatorial active-site saturation test (CAST) together with sequence conservatism analysis was applied to engineer the AhLTA toward improved Cβ stereoselectivity. The optical mutant Y314R could asymmetrically synthesize l-threo-4-methylsulfonylphenylserine with 81 % diastereomeric excess (de), which is 23 % higher than wild-type AhLTA. Molecular dynamic (MD) simulations revealed that the mechanism for the improvement in Cβ stereoselectivity of Y314R is due to the acylamino group of residues Arg314 controlling the orientation of substrate 4-methylsulfonyl benzaldehyde (1 a) in the active pocket by directed interaction with the methylsulfonyl group; this leads to asymmetric synthesis of l-threo-4-methylsulfonylphenylserine. The success in this study demonstrates that direct control of substrates in an active pocket is an attract strategy to address the Cβ stereoselectivity problem of LTA and contribute to the industrial application of LTA.

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

l-Threonine aldolase (LTA) is an attractive tool in organic chemistry for catalyzing the formation of β-hydroxy-α-amino acids with two chiral centers. The enzyme has a strict selectivity for Cα of ...

High-Throughput Screening Method for Directed Evolution and Characterization of Aldol Activity of D-Threonine Aldolase.

A rapid and reliable method for the determination of aldol condensation activity of threonine aldolases (TAs) toward aldehydes and glycine was developed. This 2,4-dinitrophenylhydrazine (DNPH) method has high sensitivity and low background disturbance and can be spectrophotometrically measured for high-throughput screening and characterization of TAs. For 4-methylsulfonyl benzaldehyde (MSB), the maximum absorbance peak was observed at around 485nm. Site-directed saturation mutagenesis libraries of D-threonine aldolase from Alcaligenes xylosoxidans CGMCC 1.4257 (AxDTA) was constructed and screened with this DNPH method for increased aldol activity toward MSB. Two beneficial variants AxDTAD321C and AxDTAN101G were identified. Substrate specificity of AxDTA and variants toward nineteen aldehydes with different substituents was facilely characterized employing this DNPH method. Furthermore, AxDTA variants displayed enhanced catalytic performance and selectivity in aldol reaction. Consequently, our study provides a rapid screening and characterization method for TAs with potential applications in preparation of chiral β-hydroxy-α-amino acids.

Construction and Application of PLP Self-sufficient Biocatalysis System for Threonine Aldolase

A number of organic synthesis involve threonine aldolase (TA), a pyridoxal phosphate (PLP)-dependent enzyme. Although the addition of exogenous PLP is necessary for the reactions, it increases the cost and complicates the purification of the product. This work constructed a PLP self-sufficient biocatalysis system for TA, which included an improvement of the intracellular PLP level and co-immobilization of TA with PLP. Engineered strain BL-ST was constructed by introducing PLP synthase PdxS/T to Escherichia coli BL21(ED3). The intracellular PLP concentration of the strain increased approximately fivefold to 48.5 μmol/gDCW. l-TA, from Bacillus nealsonii (BnLTA), was co-expressed in the strain BL-ST with PdxS/T, resulting in the engineered strain BL-BnLTA-ST. Compared with the control strain BL-BnLTA (254.1 U/L), the enzyme activity of the strain BL-BnLTA-ST reached 1518.4 U/L without the addition of exogenous PLP. An efficient co-immobilization system was then designed. The epoxy resin LX-1000HFA wrapped by polyethyleneimine (PEI) acted as a carrier to immobilize the crude enzyme solution of the strain BL-BnLTA-ST mixed with an extra 100 μM of exogenous PLP, resulting in the catalyst HFAPEI-BnLTA-STPLP 100. HFAPEI-BnLTA-STPLP 100 exhibited a half-life of approximately 450 h, and the application of the catalyst in the continuous biosynthesis of 3-[4-(methylsulfonyl) phenyl] serine had more than 180 batch reactions (>60%conv) without the extra addition of exogenous PLP. The excellent compatibility and stability of the system were further confirmed by other TAs. This work introduced a PLP self-sufficient biocatalysis system that can reduce the cost of PLP and contribute to the industrial application of TA. In addition, the system may also be applied in other PLP-dependent enzymes.

Construction of a Highly Diastereoselective Aldol Reaction System with l-Threonine Aldolase by Computer-Assisted Rational Molecular Modification and Medium Engineering.

Diastereoselectivity of l-threonine aldolase (LTA) was determined by paths of aldehydes attacking a pyridoxal phosphate-glycine complex. Thus, strategies of enhancing the syn path and blocking the anti path were performed to modify LTA. A mutant (Y31H/N305R) was constructed with a substrate preference increase from 3.32 to 42.04. Medium engineering was investigated. Consequently, the de value of l-syn-3-[4-(methylsulfonyl)phenylserine] reached 93.1% (87.2%conv). The study clarified the factors affecting diastereoselectivity of LTA and provided a theorem for rational modification of LTA's diastereoselectivity.

In silico drug target identification and pharmacophore mapping for Leishmania donovani based on metabolic pathways

Leishmania donovani is a protozoan parasite which causes very lethal disease called as visceral leishmaniasis. It is the second killer parasitic disease after malaria. It is transmitted by female sandfly and reported to be spread in 88 countries causes 20,000-30,000 death each year. It is the most neglected tropical disease in terms of drug discovery and development and till now, there is no specific vaccine or drug. We analysed the metabolic pathway of this parasite for identifying potential drug target. The essential node (gene) which is non-homologous to human in the metabolic pathway were considered for network reconstruction. Reconstructed network analysis reveals five drug targets namely: threonine aldolase, Acetyl-CoA acyltransferase, pyruvate orthophosphate dikinase, ATP-binding cassette and P-glycoprotein. These targets are efficient and specific for treating Leishmania donovani parasite. Their pharmacophore is designed and docking studies reveals the action of pharmacophore on these drug targets.

In silico drug target identification and pharmacophore mapping for Leishmania donovani based on metabolic pathways

Leishmania donovani is a protozoan parasite which causes very lethal disease called as visceral leishmaniasis. It is the second killer parasitic disease after malaria. It is transmitted by female sandfly and reported to be spread in 88 countries causes 20,000-30,000 death each year. It is the most neglected tropical disease in terms of drug discovery and development and till now, there is no specific vaccine or drug. We analysed the metabolic pathway of this parasite for identifying potential drug target. The essential node (gene) which is non-homologous to human in the metabolic pathway were considered for network reconstruction. Reconstructed network analysis reveals five drug targets namely: threonine aldolase, Acetyl-CoA acyltransferase, pyruvate orthophosphate dikinase, ATP-binding cassette and P-glycoprotein. These targets are efficient and specific for treating Leishmania donovani parasite. Their pharmacophore is designed and docking studies reveals the action of pharmacophore on these drug targets.

Identification and application of threonine aldolase for synthesis of valuable α-amino, β-hydroxy-building blocks

Chiral β-hydroxy α-amino acid structural motifs are interesting and common synthons present in multiple APIs and drug candidates. To access these chiral building blocks either multistep chemical syntheses are required or the application of threonine aldolases, which catalyze aldol reactions between an aldehyde and glycine. Bioinformatics tools have been utilized to identify the gene encoding threonine aldolase from Vanrija humicola and subsequent preparation of its recombinant version from E. coli fermentation. We planned to implement this enzyme as a key step to access the synthesis of our target API. Beyond this specific application, the aldolase was purified, characterized and the substrate scope of this enzyme further investigated. A number of enzymatic reactions were scaled-up and the products recovered to assess the diastereoselectivity and scalability of this asymmetric synthetic approach towards β-hydroxy α-amino acid chiral building blocks.

Dynamic Kinetic Resolution for Asymmetric Synthesis of L‐Noncanonical Amino Acids from D‐Ser Using Tryptophan Synthase and Alanine Racemase

L‐Ser is often used to synthesize some significant l‐noncanonical α‐amino acids(l‐ncAAs), which are the prevalent intermediates and precursors for functional synthetic compounds. In this study, threonine aldolase from Escherichia coli k‐12 MG1655 has been used to synthesize l‐Ser. In contrast to the maximum catalytic capacity (20 g/L) for l‐threonine aldolase(LTA), d‐Ser was synthesized with high yield (240 g/L) from cheap Gly and paraformaldehyde using d‐threonine aldolase (DTA) from Arthrobacter sp ATCC. In order to fully utilize d‐Ser and expand the resource of l‐Ser, a dynamic kinetic resolution system was constructed to convert d/dl‐Ser to l‐Ser through combining alanine racemase (Alr) from Bacillus subtilis with l‐tryptophan synthase (TrpS) from Escherichia coli k‐12 MG1655, and l‐ncAAs including l‐Trp and l‐Cys derivatives were synthesized with excellent enantioselectivity and in high yields. The results indicated l‐ncAAs could be efficiently synthesized from d‐Ser using this original and green dynamic kinetic resolution system, and the reliable l‐Ser resource has been established from simple and achiral substrates.

Improving and Inverting Cβ-Stereoselectivity of Threonine Aldolase via Substrate-Binding-Guided Mutagenesis and a Stepwise Visual Screening

Threonine aldolase (TA)-catalyzed aldol condensation is a powerful tool for C–C bond formation under mild conditions, but the low Cβ-stereoselectivity has hampered its wide application. A stepwise ...

Progress in using threonine aldolases for preparative synthesis

Three threonine aldolases (TAs) were cloned and overexpressed in Escherichia coli (Aeromonas jandaeil-allo-threonine aldolase, E. colil-threonine aldolase and Thermotoga maritimal-allo-threonine aldolase). A Design of Experiments strategy was used to identify optimal reaction conditions for each enzyme. These conditions were used to characterize the substrate- and stereoselectivity of each TA toward a panel of aldehyde acceptors. In general, the A. jandaei TA performed best, and six representative conversions were scaled up 10-fold in order to develop downstream steps for product isolation. One key improvement was to treat the crude reaction product with Bacillus subtilis glycine oxidase, which eliminated residual starting material and significantly simplified product isolation. NMR studies were used to identify the major and minor diastereomers from the preparative-scale reactions and the absolute configurations for three representative cases.

Application of Threonine Aldolases for the Asymmetric Synthesis of α-Quaternary α-Amino Acids.

We report the synthesis of diverse β‐hydroxy‐α,α‐dialkyl‐α‐amino acids with perfect stereoselectivity for the α‐quaternary center through the action of l‐ and d‐specific threonine aldolases. A wide variety of aliphatic and aromatic aldehydes were accepted by the enzymes and conversions up to >80 % were obtained. In the case of d‐selective threonine aldolase from Pseudomonas sp., generally higher diastereoselectivities were observed. The applicability of the protocol was demonstrated by performing enzymatic reactions on preparative scale. Using the d‐threonine aldolase from Pseudomonas sp., (2R,3S)‐2‐amino‐3‐(2‐fluorophenyl)‐3‐hydroxy‐2‐methylpropanoic acid was generated in preparative amounts in one step with a diastereomeric ratio >100 favoring the syn‐product. A Birch‐type reduction enabled the reductive removal of the β‐hydroxy group from (2S)‐2‐amino‐3‐hydroxy‐2‐methyl‐3‐phenylpropanoic acid to generate enantiopure l‐α‐methyl‐phenylalanine via a two‐step chemo‐enzymatic transformation.

Bioinformatic analysis of the fold type I PLP-dependent enzymes reveals determinants of reaction specificity in l-threonine aldolase from Aeromonas jandaei.

Understanding the role of specific amino acid residues in the molecular mechanism of a protein's function is one of the most challenging problems in modern biology. A systematic bioinformatic analysis of protein families and superfamilies can help in the study of structure–function relationships and in the design of improved variants of enzymes/proteins, but represents a methodological challenge. The pyridoxal‐5′‐phosphate (PLP)‐dependent enzymes are catalytically diverse and include the aspartate aminotransferase superfamily which implements a common structural framework known as type fold I. In this work, the recently developed bioinformatic online methods Mustguseal and Zebra were used to collect and study a large representative set of the aspartate aminotransferase superfamily with high structural, but low sequence similarity to l‐threonine aldolase from Aeromonas jandaei (LTAaj), in order to identify conserved positions that provide general properties in the superfamily, and to reveal family‐specific positions (FSPs) responsible for functional diversity. The roles of the identified residues in the catalytic mechanism and reaction specificity of LTAaj were then studied by experimental site‐directed mutagenesis and molecular modelling. It was shown that FSPs determine reaction specificity by coordinating the PLP cofactor in the enzyme's active centre, thus influencing its activation and the tautomeric equilibrium of the intermediates, which can be used as hotspots to modulate the protein's functional properties. Mutagenesis at the selected FSPs in LTAaj led to a reduction in a native catalytic activity and increased the rate of promiscuous reactions. The results provide insight into the structural basis of catalytic promiscuity of the PLP‐dependent enzymes and demonstrate the potential of bioinformatic analysis in studying structure–function relationship in protein superfamilies.

A comprehensive assessment of the biosynthetic pathways of ascorbate, α-tocopherol and free amino acids in Euglena gracilis var. saccharophila

Euglena gracilis produces several important health-enhancing metabolites including ascorbate, α-tocopherol and free amino acids (faa). The yield of metabolites is dependent on the strain of E. gracilis and the metabolic growth condition. Here we investigated the effects of photoautotrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on the synthesis of ascorbate, α-tocopherol and faa in E. gracilis var. saccharophila, using label-free shotgun proteomics, and metabolite analysis using colourimetric assay, high-performance and ultra-performance liquid chromatography (HPLC/UPLC). PT cultivation resulted in the production of more antioxidants (up to 4.13mgg−1 ascorbate and 2.52mgg−1 α-tocopherol) than the MT and HT growth conditions (up to 0.97 and 0.50mgg−1 ascorbate, and 1.40 and 0.21mgg−1 α-tocopherol, respectively). The relative abundance of several faa varied between mid-log and initial stationary growth phases, but the total amount of faa remained about the same, with arginine as the most abundant amino acid. Proteomic analysis revealed a total of 3843 non-redundant proteins in E. gracilis var. saccharophila, of which 1890 were common among all cultivations. Gene ontology annotations suggested derivatisation of metabolic pathways from different organisms, such as lysine biosynthesis from fungi and serine biosynthesis from plants, while a few pathways were unique to Euglena, such as those of ascorbate and arginine. Some enzymes exhibited several isoforms that were influenced by the metabolic growth condition. For example, one of the isozymes of threonine aldolase was expressed in HT/MT cultures only, and one of the isozymes of phosphoglycerate dehydrogenase was expressed in PT cultures only. This is the first proteomic study of E. gracilis var. saccharophila, which provides a mechanistic insight into the biosynthetic pathway dynamics of primary metabolites (antioxidants and faa). This new information can serve as a framework for further development of Euglena as a producer of nutraceuticals.

13 C metabolic flux profiling of Pichia pastoris grown in aerobic batch cultures on glucose revealed high relative anabolic use of TCA cycle and limited incorporation of provided precursors of branched-chain amino acids.

Acetohydroxy acid isomeroreductase (EC 1.1.1.86), branched-chain amino acid aminotransferase (BCAT, EC 2.6.1.42), fumarase (EC 4.2.1.2), malic enzyme (EC 1.1.1.39/1.1.1.40), phosphoenolpyruvate carboxykinase (EC 4.1.1.49), pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), l-serine hydroxymethyltransferase (EC 2.1.2.1), threonine aldolase (EC 4.1.2.5), threonine dehydratase (EC 4.3.1.19); transketolase (EC 2.2.1.1), transaldolase (EC 2.2.1.2).

A newd-threonine aldolase as a promising biocatalyst for highly stereoselective preparation of chiral aromatic β-hydroxy-α-amino acids

A newd-threonine aldolase was identified to tackle the “Cβ-stereoselectivity problem” in the enzymatic production of chiral aromatic β-hydroxy-α-amino acids.