Multistep enzyme catalysed deracemisation of 2-naphthyl alanine

Kinetic parameters of d-amino acid oxidase from R. gracilis (DAAO) towards d-2-naphthyl alanine (d-2-NAla) and of l-aspartate amino transferase (l-AAT) from Escherichia coli towards 2-naphthyl pyruvate (2-NPA) were measured. The two enzymes were then combined in a one-pot reaction in which DAAO was used to generate 2-NPA which was the substrate of l-AAT in the presence of cysteine sulphinic acid (CSA) as an amino donor. The combined reactions afforded enantiomerically pure l-2-NAla in almost quantitative yield. The extremely low water solubility of 2-NAla can be partially overcome by running the biotransformation in suspension with higher formal concentration. In these conditions multiple enzyme additions are required.     

Metabolic regulatory mechanisms and physiological roles of functional amino acids and their applications in yeast

ABSTRACT

In yeast, amino acid metabolism and its regulatory mechanisms vary under different growth environments by regulating anabolic and catabolic processes, including uptake and export, and the metabolic styles form a complicated but robust network. There is also crosstalk with various metabolic pathways, products and signal molecules. The elucidation of metabolic regulatory mechanisms and physiological roles is important fundamental research for understanding life phenomenon. In terms of industrial application, the control of amino acid composition and content is expected to contribute to an improvement in productivity, and to add to the value of fermented foods, alcoholic beverages, bioethanol, and other valuable compounds (proteins and amino acids, etc.). This review article mainly describes our research in constructing yeast strains with high functionality, focused on the metabolic regulatory mechanisms and physiological roles of “functional amino acids”, such as l-proline, l-arginine, l-leucine, l-valine, l-cysteine, and l-methionine, found in yeast.     

Forced evolution of Escherichia coli cells with the ability to effectively utilize non-natural amino acids l-tert-leucine,l-norleucine and γ-methyl-l-leucine

Abstract

In an attempt to develop a novel biocatalyst able to efficiently catalyse the synthesis of non-natural amino acids, Escherichia coli TG1 was treated with 10 mM NaNO₂ and then cultured in selective medium supplemented with 20 mM l-tert-leucine. Each culture was grown for 2 weeks and then subcultured into fresh medium with successive decreases of l-tert-leucine concentration at each transfer to a final value of 0.5 mM. The adapted cells resulting from this forced evolution procedure were able to grow in minimal medium with 0.1 mM l-tert-leucine as sole nitrogen source. Both HPLC and TLC verified progressive removal of l-tert-leucine from the medium during bacterial growth. Further studies revealed that the adapted cells metabolized l-tert-leucine by transamination, removing the amino group but leaving the carbon skeleton of the corresponding 2-oxoacid intact. Despite the mutagenesis, when the four obvious candidate amino acid aminotransferase genes were cloned and sequenced, there was no change in these structural genes. The activity of the adapted cells with l-tert-leucine is apparently attributable to the wild-type branched-chain amino acid aminotransferase (IlvAT), presumably expressed at higher levels as a result of a regulatory mutation. With the isolate I-4, the resting cells transaminate l-tert-leucine, l-norleucine, l-norvaline, γ-methyl-l-leucine and dl-homophenylalanine as effectively as does the crude extract. These evolved cells may be useful for synthesizing non-natural amino acids for the pharmaceutical industry. In addition, the adapted cells can also catalyse transamination of naturally occurring hydrophobic amino acids.     

R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport

The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)—a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport.     

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.     

Asymmetric synthesis of l-6-hydroxynorleucine from 2-keto-6-hydroxyhexanoic acid using a branched-chain aminotransferase

Abstract

l-6-Hydroxynorleucine was synthesized from 2-keto-6-hydroxyhexanoic acid using branched-chain aminotransferase from Escherichia coli with l-glutamate as an amino donor. Since the branched-chain aminotransferase was severely inhibited by 2-ketoglutarate, the branched-chain aminotransferase reaction was coupled with aspartate aminotransferase and pyruvate decarboxylase. Aspartate aminotransferase converted the inhibitory 2-ketoglutarate back to l-glutamate by using l-aspartate as an amino donor. On the other hand, pyruvate decarboxylase further shifted the reaction equilibrium towards l-6-hydroxynorleucine through decarboxylation of pyruvate to acetaldehyde. The concerted action of the three enzymes significantly enhanced the yield compared to that of branched-chain aminotransferase alone. In the coupled reaction, 90.2 mM l-6-hydroxynorleucine (> 99% ee) was produced from 100 mM 2-keto-6-hydroxyhexanoic acid, whereas in a single branched-chain aminotransferase reaction only 22.5 mM l-6-hydroxynorleucine (> 99% ee) was produced.     

Separation of l-Leucine-pyruvate and l-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.

The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.     

Partial Purification and Some Properties of 7-Keto-8-aminopelargonic Acid Synthetase,an Enzyme Involved in Biotin Biosynthesis

7-Keto-8-aminopelargonic acid synthetase (KAPA synthetase) which catalyzes the formation of KAPA from pimelyl CoA and l-alanine, and is involved in biotin biosynthesis, was partially purified from a cell-free extract of Bacillus sphaericus by a procedure involving ammonium sulfate fraction ation, protamine treatment, and DEAE-cellulose column chromatography. The reaction product was bioautographically confirmed to be KAPA. Some properties of the enzyme were also investigated. Among the amino acids, only l-alanine was active as a substrate, condensing with pimelyl CoA, The reaction required pyridoxal phosphate but the other vitamin B₆ compounds were inert. Typical inhibitors of pyridoxal phosphate enzymes showed marked inhibition to the reaction. Various amino acids such as l-cysteine, glycine, d-alanine, l-serine, l-histidine, and d-histidine were also found to be inhibitory.     

Some Properties of Three Proteolytic Enzymes Excreted by Bacillus cereus Kp 931

The crude enzyme preparation obtained from culture media of Bacillus cereus Kp 931 was fractionated into three active fractions by Sephadex G-100 gel filtration. These three enzymes had pH optima at between 10.5 and 11.0. One of them, the largest molecular weight species, the enzyme I, was purified extensively. The enzyme catalyzes the release of a number of free amino acids from casein. Large amounts of l-alanine and l-glutamic acid and small amounts of l-leucine, l-serine, glycine, l-cysteic acid and l-arginine were released from oxidized insulin B-chain by the action of the purified enzyme I. It is also suggested that the other two enzymes, II and III, belong to so-called bacterial proteninases.     

Taurine Levels in Some Tissues of Yellowtail (Seriola quinqueradiata) and Mackerel (Scomber japonicus)

The mechanism of stereospecific production of l-amino acids from the corresponding 5-substituted hydantoins by Bacillus brevis AJ-12299 was studied. The enzymes involved in the reaction were partially purified by DEAE-Toyopearl 650M column chromatography and their properties were investigated. The conversion of dl-5-substituted hydantoins to the corresponding l-amino acids consisted of the following two successive reactions. The first step was the ring-opening hydrolysis to N-carbamoyl amino acids catalyzed by an ATP dependent l-5-substituted hydantoin hydrolase. This reaction was stereospecific and the N-carbamoyl amino acid produced was exclusively the l-form. N-Carbamoyl-l-amino acid was also produced from the d-form of 5-substituted hydantoin, which suggests that spontaneous racemization occurred in the reaction mixture. In the second step, N-carbamoyl-l-amino acid was hydrolyzed to l-amino acid by an N-carbamoyl-l-amino acid hydrolase, which was also an l-specific enzyme. The ATP dependency of the l-5-substituted hydantoin hydrolase was supposed to be the limiting factor in the production of l-amino acids from the corresponding 5-substituted hydantoins by this bacterium.     

Structure,function, and regulation of enzymes involved in amino acid metabolism of bacteria and archaea

Amino acids are essential components in all organisms because they are building blocks of proteins. They are also produced industrially and used for various purposes. For example, L-glutamate is used as the component of “umami” taste and lysine has been used as livestock feed. Recently, many kinds of amino acids have attracted attention as biological regulators and are used for a healthy life. Thus, to clarify the mechanism of how amino acids are biosynthesized and how they work as biological regulators will lead to further effective utilization of them. Here, I review the leucine-induced-allosteric activation of glutamate dehydrogenase (GDH) from Thermus thermophilus and the relationship with the allosteric regulation of GDH from mammals. Next, I describe structural insights into the efficient production of L-glutamate by GDH from an excellent L-glutamate producer, Corynebacterium glutamicum. Finally, I review the structural biology of lysine biosynthesis of thermophilic bacterium and archaea.     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits

ABSTRACT

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants.

Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3?,5?-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase     

The Biosynthetic Control in Aromatic Amino Acid Producing Mutants of Corynebacterium glutamicum

Regulatory properties of the enzymes involved in aromatic amino acid biosynthesis in the mutant of Corynebacterium glutamicum which produces a large amount of aromatic amino acids were examined. A phenylalanine auxotrophic l-tyrosine producer, pr-20, had a 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthetase released from the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a two-fold derepressed chorismate mutase. A pair of l-phenylalanine and l-tyrosine still strongly inhibited the chorismate mutase activity, though the enzyme was partially released from the inhibition by l-phenylalanine alone. A tyrosine auxotrophic l-phenylalanine producer, PFP-19-31, had a DAHP synthetase sensitive to the feedback inhibition by l-phenylalanine, l-tyrosine and l-tryptophan and had a prephenate dehydratase and a chorismate mutase both partially released from the feedback inhibition by l-phenylalanine. The mutant produced a large amount of prephenate as well as l-phenylalanine. A phenylalanine and tyrosine double auxotrophic l-tryptophan producer, Px-115-97, had an anthranilate synthetase partially released from the feedback inhibition by l-tryptophan and had a DAHP synthetase sensitive to the feedback inhibition. These data explained the mechanism of the production of aromatic amino acids by these mutants and supported the in vivo functioning of the control mechanisms of aromatic amino acid biosynthesis in C. glutamicum previously elucidated in vitro experiments.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain l-amino acids, l-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for l-leucine, and the k_cat/Km value for l-leucine was higher than that for l-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD ⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5′-phosphate. d-Enantiomers of the substrate amino acids competitively inhibited the oxidation of l-valine.     

Mechanism of Asymmetric Production of d-Amino Acids from the Corresponding Hydantoins by Pseudomonas sp.

The mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 was examined by investigating the properties of the enzymes involved in the hydrolysis of dl-5-substituted hydantoins. The enzymatic production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. AJ-11220 involved the following two successive reactions; the d-isomer specific hydrolysis, i.e., the ring opening of d-5-substituted hydantoins to d-form N-carbamyl amino acids by an enzyme, d-hydantoin hydrolase (d-HYD hydrolase), followed by the d-isomer specific hydrolysis, i.e., the cleavage of N-carbamyl-d-amino acids to d-amino acids by an enzyme, N-carbamyl-d-amino acid hydrolase (d-NCA hydrolase).

l-5-Substituted hydantoins not hydrolyzed by d-HYD hydrolase were converted to d-form 5- substituted hydantoins through spontaneous racemization under the enzymatic reaction conditions.

It was proposed that almost all of the dl-5-substituted hydantoins were stoichiometrically and directly converted to the corresponding d-amino acids through the successive reactions of d-HYD hydrolase and d-NCA hydrolase in parrallel with the spontaneous racemization of l-5-substituted hydantoins to those of dl-form.     

Synthesis of γ-Glutamylpeptides by γ-Glutamylcysteine Synthetase from Proteus mirabilis

Syntheses of various γ-glutamylpeptides were examined taking use of the highly purified γ-glutamylcysteine synthetase from Proteus mirabilis. The accumulation of each peptide was measured after long time incubation, and good formation was observed in the synthesis of peptides of following amino acids, l-cysteine, l-α-aminobutyrate, l-serine, l-homoserine, glycine, l-alanine, l-norvaline, l-lysine, l-threonine, taurine and l-valine. Peptide syntheses were confirmed by analyses of the component amino acids, after hydrolysis of the peptides.

The structure of the glutamylpeptides, especially the peptide-linkage at the γ-carbonyl residue of l-glutamate, was determined by mass spectrometry of the N-trifluoroacetyl methylester derivatives of the glutamylpeptides. Enzymatic synthesis of γ-glutamyl-l-α-aminobutyrate was also confirmed by PMR spectrometry in the comparison with chemically synthesized compound.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.