Amino Donor Research Articles

Biosynthesis of the Essential Respiratory Cofactor Ubiquinone from Phenylalanine in Plants

The prenylated benzoquinone coenzyme Q or ubiquinone is a vital electron and proton carrier in the respiratory chain of mitochondria and some bacteria (Nowicka and Kruk, 2010). Due to its highly hydrophobic nature, ubiquinone can freely migrate in mitochondrial inner membrane carrying one or two electrons from either NADH dehydrogenase (complex I) or succinate dehydrogenase (complex II) to cytochrome bc1 complex (complex III). In plants, ubiquinone can additionally be reduced and oxidized by alternative NAD(P)H dehydrogenases and alternative oxidase, respectively.

The archaic chaperone-usher pathways may depend on donor strand exchange for intersubunit interactions.

Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated.

Heterologous pathway for the production of l-phenylglycine from glucose by E. coli

The aproteinogenic amino acid l-phenylglycine (l-Phg) is an important side chain building block for the preparation of several antibiotics and taxol. To biosynthesis l-Phg from glucose, an engineered Escherichia coli containing l-Phg synthetic genes was firstly developed by an l-phenylalanine producing chassis supplying phenylpyruvate. The enzymes HmaS (l-4-hydroxymandelate synthase), Hmo (l-4-hydroxymandelate oxidase) and HpgT (l-4-hydroxyphenylglycine transaminase) from Amycolatopsis orientalis as well as Streptomyces coelicolor were heterologously expressed in E. coli and purified to evaluate their abilities on l-Phg formation. HpgT conversing phenylglyoxylate to l-Phg uses an unusual amino donor l-phenylalanine, which releases another phenylpyruvate as the substrate of HmaS. Thus, a recycle reaction was developed to maximize the utilization of precursor phenylpyruvate. To amplify the accumulation of l-Phg, the effects of attenuating l-phenylalanine transamination was investigated. After deletion of tyrB and aspC, l-Phg yield increased by 12.6-fold. The limiting step in the l-Phg biosynthesis was also studied; the l-Phg yield was further improved by 14.9-fold after enhancing hmaS expression. Finally, by optimizing expression of hmaS, hmo and hpgT and attenuation of l-phenylalanine transamination, the l-Phg yield was increased by 224-fold comparing with the original strain.

ChemInform Abstract: Aminolysis of Aryl Ester Using Tertiary Amine as Amino Donor via C—O and C—N Bond Activations.

An aminolysis reaction between various aryl esters and inert tertiary amines by C–O and C–N bond activations has been developed for the selective synthesis of a broad scope of tertiary amides under neutral and mild conditions. The mechanism may undergo the two key steps of oxidative addition of acyl C–O bond in parent ester and C–N bond cleavage of tertiary amine via an iminium-type intermediate.

Immobilization of (R)- and (S)-amine transaminases on chitosan support and their application for amine synthesis using isopropylamine as donor

Transaminases from Aspergillus fumigatus ((R)-selective, AspFum), Ruegeria pomeroyi ((S)-selective, 3HMU) and Rhodobacter sphaeroides 2.4.1 ((S)-selective, 3I5T) were immobilized on chitosan with specific activities of 99, 157, and 163U/g and acceptable yields (54, 21, and 23%, respectively) for glutaraldehyde (GA) immobilization. Besides GA, also divinylsulfone was used as linker molecule leading to a similar efficient immobilization for two enzymes, GibZea and NeoFis, whereas GA was superior in the other cases. Storage of the GA-immobilized enzymes for one month resulted in increased relative activities between 120 and 180%. The thermal stability was improved, especially for the GA-immobilized AspFum compared to the free enzyme after incubation for 4h at 60°C (10% vs. 235% residual activity). Especially after incubation of AspFum (free or immobilized) for 2h at 50°C a strongly increased activity was observed (up to 359% of the initial activity). This effect was studied in more detail, revealing that one heat activation prior and one after immobilization increased the overall immobilization efficiency. Recycling of the immobilized ATAs resulted only in a small reduction of activity after four batches. Asymmetric synthesis of (R)- or (S)-1-methyl-3-phenylpropylamine from the prostereogenic ketone using isopropylamine (IPA) as amino donor was applied with conversions up to 50% (AspFum) or 75% (3HMU). Except for NeoFis, all immobilized ATAs showed higher conversions compared to the free enzyme.

Redox self-sufficient whole cell biotransformation for amination of alcohols

Whole cell biotransformation is an upcoming tool to replace common chemical routes for functionalization and modification of desired molecules. In the approach presented here the production of various non-natural (di)amines was realized using the designed whole cell biocatalyst Escherichia coli W3110/pTrc99A-ald-adh-ta with plasmid-borne overexpression of genes for an l-alanine dehydrogenase, an alcohol dehydrogenase and a transaminase. Cascading alcohol oxidation with l-alanine dependent transamination and l-alanine dehydrogenase allowed for redox self-sufficient conversion of alcohols to the corresponding amines. The supplementation of the corresponding (di)alcohol precursors as well as amino group donor l-alanine and ammonium chloride were sufficient for amination and redox cofactor recycling in a resting buffer system. The addition of the transaminase cofactor pyridoxal-phosphate and the alcohol dehydrogenase cofactor NAD+ was not necessary to obtain complete conversion. Secondary and cyclic alcohols, for example, 2-hexanol and cyclohexanol were not aminated. However, efficient redox self-sufficient amination of aliphatic and aromatic (di)alcohols in vivo was achieved with 1-hexanol, 1,10-decanediol and benzylalcohol being aminated best.

Evidence that glutamine transaminase and omega-amidase potentially act in tandem to close the methionine salvage cycle in bacteria and plants

S-Adenosylmethionine is converted enzymatically and non-enzymatically to methylthioadenosine, which is recycled to methionine (Met) via a salvage pathway. In plants and bacteria, enzymes for all steps in this pathway are known except the last: transamination of α-ketomethylthiobutyrate to give Met. In mammals, glutamine transaminase K (GTK) and ω-amidase (ω-Am) are thought to act in tandem to execute this step, with GTK forming α-ketoglutaramate, which ω-Am hydrolyzes. Comparative genomics indicated that GTK and ω-Am could function likewise in plants and bacteria because genes encoding GTK and ω-Am homologs (i) co-express with the Met salvage gene 5-methylthioribose kinase in Arabidopsis, and (ii) cluster on the chromosome with each other and with Met salvage genes in diverse bacteria. Consistent with this possibility, tomato, maize, and Bacillus subtilis GTK and ω-Am homologs had the predicted activities: GTK was specific for glutamine as amino donor and strongly preferred α-ketomethylthiobutyrate as amino acceptor, and ω-Am strongly preferred α-ketoglutaramate. Also consistent with this possibility, plant GTK and ω-Am were localized to the cytosol, where the Met salvage pathway resides, as well as to organelles. This multiple targeting was shown to result from use of alternative start codons. In B. subtilis, ablating GTK or ω-Am had a modest but significant inhibitory effect on growth on 5-methylthioribose as sole sulfur source. Collectively, these data indicate that while GTK, coupled with ω-Am, is positioned to support significant Met salvage flux in plants and bacteria, it can probably be replaced by other aminotransferases.

Asparagine Depletion Potentiates the Cytotoxic Effect of Chemotherapy against Brain Tumors

Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner--as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy.

Aminotransferase activity of a novel aminoacyl‐tRNA synthetase appended domain (751.3)

Aminoacyl‐tRNA synthetases (AARSs) are key enzymes in protein biosynthesis, responsible for attaching amino acids to the 3ʹ‐end of cognate tRNAs. The AARSs are modular proteins, with separate polypeptide domains responsible for tRNA binding and catalysis; additional domains on some AARSs contribute oligomerization, localization, and editing functions. The opportunistic pathogen Mycoplasms penetrans expresses an unusually long version of methionyl‐tRNA synthetase (MetRS) that contains an extra N‐terminal domain with sequence homology to class V aminotransferases. We anticipated that this domain carries out pyridoxal phosphate (PLP)‐dependent aminotransferase activity, possibly to modify Met‐tRNA(Met) for enhanced pathogenicity.We overexpressed and purified M. penetrans MetRS (MpMetRS) and used the amino‐reactive aromatic dialdehyde naphthalene‐2,3‐dicarboxaldehyde followed by HPLC to analyze enzyme activity in vitro. Initial characterization confirms the proposed aminotransferase activity, with several amino acids and α‐keto acids serving as amino group donors and acceptors, respectively. Substitution of amino acids in the transferase and synthetase catalytic sites indicate that these activities are independent. Ultimately we seek to identify the cellular substrates and implications of the MpMetRS aminotransferase activity.Grant Funding Source: Supported by the National Science Foundation

ChemInform Abstract: Expanding Dynamic Kinetic Protocols: Transaminase‐Catalyzed Synthesis of α‐Substituted β‐Amino Ester Derivatives.

AbstractSeveral enantiopure α‐alkylated β‐amino esters (II)/(III) are obtained via dynamic kinetic resolution employing a kit of transaminases as biocatalysts and isopropylamine as amino donor.

Expression, purification, and characterization of mouse glycine N-acyltransferase in Escherichia coli

Glycine N-acyltransferase (GLYAT) is a phase II metabolic detoxification enzyme for exogenous (xenobiotic) and endogenous carboxylic acids; consisting of fatty acids, benzoic acid, and salicylic acid. GLYAT catalyzes the formation of hippurate (N-benzoylglycine) from the corresponding glycine and benzoyl-CoA. Herein, we report the successful expression, purification, and characterization of recombinant mouse GLYAT (mGLYAT). A 34kDa mGLYAT protein was expressed in Escherichia coli and purified to homogeneity by nickel affinity chromatography to a final yield of 2.5mg/L culture. Characterization for both amino donors and amino acceptors were completed, with glycine serving as the best amino donor substrate, (kcat/Km)app=(5.2±0.20)×102M−1s−1, and benzoyl-CoA serving as the best the amino acceptor substrate, (kcat/Km)app=(4.5±0.27)×105M−1s−1. Our data demonstrate that mGLYAT will catalyzed the chain length specific (C2–C6) formation of N-acylglycines. The steady-state kinetic constants determined for recombinant mGLYAT for the substrates benzoyl-CoA and glycine, were shown to be consistent with other reported species (rat, human, bovine, ovine, and rhesus monkey). The successful recombinant expression and purification of mGLYAT can lead to solve unanswered questions associated with this enzyme, consisting of what is the chemical mechanism and what catalytic residues are essential for the how this phase II metabolic detoxification enzyme conjugates glycine to xenobiotic and endogenous carboxylic acids.

Three Different Classes of Aminotransferases Evolved Prephenate Aminotransferase Functionality in Arogenate-competent Microorganisms

The aromatic amino acids phenylalanine and tyrosine represent essential sources of high value natural aromatic compounds for human health and industry. Depending on the organism, alternative routes exist for their synthesis. Phenylalanine and tyrosine are synthesized either via phenylpyruvate/4-hydroxyphenylpyruvate or via arogenate. In arogenate-competent microorganisms, an aminotransferase is required for the transamination of prephenate into arogenate, but the identity of the genes is still unknown. We present here the first identification of prephenate aminotransferases (PATs) in seven arogenate-competent microorganisms and the discovery that PAT activity is provided by three different classes of aminotransferase, which belong to two different fold types of pyridoxal phosphate enzymes: an aspartate aminotransferase subgroup 1β in tested α- and β-proteobacteria, a branched-chain aminotransferase in tested cyanobacteria, and an N-succinyldiaminopimelate aminotransferase in tested actinobacteria and in the β-proteobacterium Nitrosomonas europaea. Recombinant PAT enzymes exhibit high activity toward prephenate, indicating that the corresponding genes encode bona fide PAT. PAT functionality was acquired without other modification of substrate specificity and is not a general catalytic property of the three classes of aminotransferases.

Effects of iptycene scaffolds on the photoluminescence of N, N-dimethylaminobenzonitrile and its analogues

To understand the effect of iptycene scaffolds on the locally excited (LE) and intramolecular charge transfer (ICT) fluorescence of aminobenzonitriles, a series of triptycene and pentiptycene derivatives were synthesized and their molecular structures and photophysical properties were characterized and compared with the parent phenylene systems, 4-(N-methylamino)benzonitrile (MABN), 4-(N,N-dimethylamino)benzonitrile (DMABN), and 4-(N-phenylamino)benzonitrile (PABN). The iptycene effect does not change the nature of the fluorescing states for each amino donor system, i.e., the MA, PA, and DMA series display LE-only, ICT-only, and LE-ICT dual fluorescences, respectively. However, the iptycene scaffolds impose a significant modification of the absorption and emission spectra, fluorescence quantum efficiency and lifetimes, and the interplay of LE and ICT states. The observed iptycene effect has been discussed with three factors: (1) steric effect on increasing the amino twist angle, (2) steric shielding of solvation to the aminobenzonitrile core, and (3) hyperconjugation interactions of the aminobenzonitrile core with the peripheral phenylene groups of iptycene.

Crystal Structure of an (R)-Selective ω-Transaminase from Aspergillus terreus

Chiral amines are important building blocks for the synthesis of pharmaceutical products, fine chemicals, and agrochemicals. ω-Transaminases are able to directly synthesize enantiopure chiral amines by catalysing the transfer of an amino group from a primary amino donor to a carbonyl acceptor with pyridoxal 5′-phosphate (PLP) as cofactor. In nature, (S)-selective amine transaminases are more abundant than the (R)-selective enzymes, and therefore more information concerning their structures is available. Here, we present the crystal structure of an (R)-ω-transaminase from Aspergillus terreus determined by X-ray crystallography at a resolution of 1.6 Å. The structure of the protein is a homodimer that displays the typical class IV fold of PLP-dependent aminotransferases. The PLP-cofactor observed in the structure is present in two states (i) covalently bound to the active site lysine (the internal aldimine form) and (ii) as substrate/product adduct (the external aldimine form) and free lysine. Docking studies revealed that (R)-transaminases follow a dual binding mode, in which the large binding pocket can harbour the bulky substituent of the amine or ketone substrate and the α-carboxylate of pyruvate or amino acids, and the small binding pocket accommodates the smaller substituent.

Transaminases Applied to the Synthesis of High Added-Value Enantiopure Amines

Critical parameters affecting the stereoselective amination of (hetero)aromatic ketones using transaminases have been studied, such as temperature, pH, substrate concentration, cosolvent, and source and percentage of amino donor, to further optimize the production of enantiopure amines using both (S)- and (R)-selective biocatalysts from commercial suppliers. Interesting enantiopure amino building blocks have been obtained, overcoming some limitations of traditional chemical synthetic methods. Representative processes were scaled up, affording halogenated and heteroaromatic amines in enantiomerically pure form and good isolated yields.

Neutral and Cationic Zinc Complexes with N- and S-Donor-Functionalized Cyclopentadienyl Ligands

The synthesis of neutral and cationic zinc cyclopentadienyl (Cp) complexes with amino and thio donor groups that are attached to the Cp ring via a side chain is reported. The amino-functionalized zincocene [ZnCp3N2] (3; Cp3N = C5Me4(CH2)3NMe2) was prepared in a salt metathesis reaction from KCp3N and ZnCl2. In the solid-state structure of 3, which was determined by single-crystal X-ray diffraction, one of the Cp3N ligands is coordinated as a chelating ligand through a ring carbon atom and through the amino group, whereas the other Cp3N ligand is bound in a monodentate mode through the Cp ring only. A reaction of 3 with ZnEt2, [ZnCp*2] (Cp* = C5Me5), and [ZnCp2N2] (2; Cp2N = C5Me4(CH2)2NMe2) gave the heteroleptic complexes [ZnEtCp3N] (4), [ZnCp*Cp3N] (5), and [ZnCp3NCp2N] (6), respectively. The incorporation of a second amino group in the same side chain led to the formation of the mononuclear zinc chlorido complex [ZnClCptmeda] (7; Cptmeda = C5Me4(CH2)2NMe(CH2)2NMe2), in which the Cptmeda ligand is bound ...

Aminolysis of Aryl Ester Using Tertiary Amine as Amino Donor via C–O and C–N Bond Activations

An aminolysis reaction between various aryl esters and inert tertiary amines by C-O and C-N bond activations has been developed for the selective synthesis of a broad scope of tertiary amides under neutral and mild conditions. The mechanism may undergo the two key steps of oxidative addition of acyl C-O bond in parent ester and C-N bond cleavage of tertiary amine via an iminium-type intermediate.

Plant amino acid-derived vitamins: biosynthesis and function

Vitamins are essential organic compounds for humans, having lost the ability to de novo synthesize them. Hence, they represent dietary requirements, which are covered by plants as the main dietary source of most vitamins (through food or livestock's feed). Most vitamins synthesized by plants present amino acids as precursors (B1, B2, B3, B5, B7, B9 and E) and are therefore linked to plant nitrogen metabolism. Amino acids play different roles in their biosynthesis and metabolism, either incorporated into the backbone of the vitamin or as amino, sulfur or one-carbon group donors. There is a high natural variation in vitamin contents in crops and its exploitation through breeding, metabolic engineering and agronomic practices can enhance their nutritional quality. While the underlying biochemical roles of vitamins as cosubstrates or cofactors are usually common for most eukaryotes, the impact of vitamins B and E in metabolism and physiology can be quite different on plants and animals. Here, we first aim at giving an overview of the biosynthesis of amino acid-derived vitamins in plants, with a particular focus on how this knowledge can be exploited to increase vitamin contents in crops. Second, we will focus on the functions of these vitamins in both plants and animals (and humans in particular), to unravel common and specific roles for vitamins in evolutionary distant organisms, in which these amino acid-derived vitamins play, however, an essential role.

An alternative pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine:phenylpyruvate aminotransferase

Phenylalanine is a vital component of proteins in all living organisms, and in plants is a precursor for thousands of additional metabolites. Animals are incapable of synthesizing phenylalanine and must primarily obtain it directly or indirectly from plants. Although plants can synthesize phenylalanine in plastids through arogenate, the contribution of an alternative pathway via phenylpyruvate, as occurs in most microbes, has not been demonstrated. Here we show that plants also utilize a microbial-like phenylpyruvate pathway to produce phenylalanine, and flux through this route is increased when the entry point to the arogenate pathway is limiting. Unexpectedly, we find the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the coordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extra-plastidial metabolism of these amino acids. This discovery uncovers another level of complexity in the plant aromatic amino acid regulatory network, unveiling new targets for metabolic engineering.

Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids

Abstract A new ( S )-enantioselective ω-transaminase (ω-TA) gene from Burkholderia vietnamiensis G4 was functionally expressed in Escherichia coli BL21 (DE3), and the purified recombinant N-terminal His-tagged ω-TA (HBV-ω-TA) had a dimeric structure with optimum pH and temperature of 8.4 and 40 °C, respectively. The enzyme showed higher activities toward aromatic amines than aliphatic amines and ( S )-1-methylbenzylamine (( S )-α-MBA) was the most active amino donor. For amino acceptor, keto acids, keto esters and aldehydes were more reactive than ketones with pyruvate ethyl ester being most active. Several chiral amines and unnatural amino acids or esters were synthesized using HBV-ω-TA as the catalyst and isopropylamine or ( S )-α-MBA as amino donor. Notably, HBV-ω-TA catalyzed the amino transfer to β-keto esters to give optically pure β-amino acid esters. In addition, glyoxylate was used as amino acceptor for the first time in the kinetic resolution of racemic amines and optically pure amines, such as ( R )-1-methylbenzylamine, ( R )-1-phenylpropylamine, ( R )-2-amino-4-phenylbutane and ( R )-1-aminotetraline, were obtained.