An Enzyme Assay Kit for GABA Quantification in Plant Tissues.

Gamma-aminobutyric acid (GABA) is an amino acid that has a role as a signaling molecule. In plants, its involvement in stress responses is widely investigated. A newly developed method of quantification of GABA is described in this chapter. The assay kit consisting of three bacterial enzymes enables easy but accurate measurement of GABA (~200 mg/mL) based on the serial enzymatic reaction leading to dye formation. The method was successfully applied to measure the GABA content in several plant tissues.

A new l-arginine oxidase engineered from l-glutamate oxidase.

The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 catalyzes the oxidative deamination of l-glutamate to produce 2-ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l-glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l-glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l-arginine. The oxidative deamination activity of LGOX to l-arginine is higher than that of l-arginine oxidase form from Pseudomonas sp. TPU 7192. X-ray crystal structure analysis revealed that the guanidino group of l-arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild-type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l-arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l-arginine by LGOX R305E, is proportional to the concentration of l-arginine in a range from 0 to 100 µM. The linear relationship is maintained around 1 µM of l-arginine. Thus, LGOX R305E is suitable for the determination of l-arginine.

Structural basis of enzyme activity regulation by the propeptide of l-lysine α-oxidase precursor from Trichoderma viride.

Harmuful proteins are usually synthesized as inactive precursors and are activated by proteolytic processing. l-Amino acid oxidase (LAAO) is a flavoenzyme that catalyzes the oxidative deamination of l-amino acid to produce a 2-oxo acid with ammonia and highly toxic hydrogen peroxide and, therefore, is expressed as a precursor. The LAAO precursor shows significant variation in size and the cleavage pattern for activation. However, the molecular mechanism of how the propeptide suppresses the enzyme activity remains unclear except for deaminating/decarboxylating Pseudomonasl-phenylalanine oxidase (PAO), which has a short N-terminal propeptide composed of 14 residues. Here we show the inactivation mechanism of the l-lysine oxidase (LysOX) precursor (prLysOX), which has a long N-terminal propeptide composed of 77 residues, based on the crystal structure at 1.97 Šresolution. The propeptide of prLysOX indirectly changes the active site structure to inhibit the enzyme activity. prLysOX retains weak enzymatic activity with strict specificity for l-lysine and shows raised activity in acidic conditions. The structures of prLysOX crystals that soaked in a solution with various concentrations of l-lysine have revealed that prLysOX can adopt two conformations; one is the inhibitory form, and the other is very similar to mature LysOX. The propeptide region of the latter form is disordered, and l-lysine is bound to the latter form. These results indicate that prLysOX uses a different strategy from PAO to suppress the enzyme activity and suggest that prLysOX can be activated quickly in response to the environmental change without proteolytic processing.

Structural basis of strict substrate recognition of l-lysine α-oxidase from Trichoderma viride.

l-Lysine oxidase (LysOX) is a FAD-dependent homodimeric enzyme that catalyzes the oxidative deamination of l-lysine to produce α-keto-ε-aminocaproate with ammonia and hydrogen peroxide. LysOX shows strict substrate specificity for l-lysine, whereas most l-amino acid oxidases (LAAOs) exhibit broad substrate specificity for l-amino acids. Previous studies of LysOX showed that overall structural similarity to the well-studied snake venom LAAOs. However, the molecular mechanism of strict specificity for l-lysine was still unclear. We here determined the structure of LysOX in complex with l-lysine at 1.7 Å resolution. The structure revealed that the hydrogen bonding network formed by D212, D315, and A440 with two water molecules is responsible for the recognition of the side chain amino group. In addition, a narrow hole formed by five hydrophobic residues in the active site contributes to strict substrate specificity. Mutation studies demonstrated that D212 and D315 are essential for l-lysine recognition, and the D212A/D315A double mutant LysOX showed different substrate specificity from LysOX. Moreover, the structural basis of the substrate specificity change has also been revealed by the structural analysis of the mutant variant and its substrate complexes. These results clearly explain the molecular mechanism of the strict specificity of LysOX and suggest that LysOX is a potential candidate for a template to design LAAOs specific to other l-amino acids.

GABA enzymatic assay kit.

We developed an enzymatic assay system enabling easy quantification of 4-aminobutyric acid (GABA). The reaction of GABA aminotransferase obtained from Streptomyces decoyicus NBRC 13977 was combined to those of the previously developed glutamate assay system using glutamate oxidase and peroxidase. The three-enzyme system allowing GABA-dependent dye formation due to the oxidative coupling between 4-aminoantipyrine and Trinder's reagent enabled accurate quantification of 0.2 - 150 mg/L GABA. A pretreatment mixture consisting of glutamate oxidase, ascorbate oxidase and catalase eliminating glutamate, ascorbate, and hydrogen peroxide, respectively, was also prepared to remove those inhibitory substances from samples. Thus, constructed assay kit was used to measure the GABA content in tomato samples. The results were almost the same as that obtained by the conventional method using liquid chromatography-tandem mass spectrometry. The kit will become a promising tool especially for the on-site measurement of GABA content in agricultural products.

Recombinant expression, molecular characterization and crystal structure of antitumor enzyme, L-lysine α-oxidase from Trichoderma viride.

L-Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L-lysine to form α-keto-ε-aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L-amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

Natural low-molecular mass organic compounds with oxidase activity as organocatalysts.

Organocatalysts, low-molecular mass organic compounds composed of nonmetallic elements, are often used in organic synthesis, but there have been no reports of organocatalysts of biological origin that function in vivo. Here, we report that actinorhodin (ACT), a natural product derived from Streptomyces coelicolor A3(2), acts as a biocatalyst. We purified ACT and assayed its catalytic activity in the oxidation of L-ascorbic acid and L-cysteine as substrates by analytical methods for enzymes. Our findings were as follows: (i) oxidation reactions producing H2O2 proceeded upon addition of ACT to the reaction mixture; (ii) ACT was not consumed during the reactions; and (iii) a small amount (catalytic amount) of ACT consumed an excess amount of the substrates. Even at room temperature, atmospheric pressure, and neutral pH, ACT showed catalytic activity in aqueous solution, and ACT exhibited substrate specificity in the oxidation reactions. These findings reveal ACT to be an organocatalyst. ACT is known to show antibiotic activity, but its mechanism of action remains unknown. On the basis of our results, we propose that ACT kills bacteria by catalyzing the production of toxic levels of H2O2. We also screened various other natural products of bacterial, plant, and animal origins and found that several of the compounds exhibited catalytic activity, suggesting that living organisms produce and use these compounds as biocatalysts in nature.

Arg305 of Streptomyces L-glutamate oxidase plays a crucial role for substrate recognition.

Recently, we have solved the crystal structure of L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 (PDB code: 2E1M), the substrate specificity of which is strict toward L-glutamate. By a docking simulation using L-glutamate and structure of LGOX, we selected three residues, Arg305, His312, and Trp564 as candidates of the residues associating with recognition of L-glutamate. The activity of LGOX toward L-glutamate was significantly reduced by substitution of selected residues with Ala. However, the enzyme, Arg305 of which was substituted with Ala, exhibited catalytic activity toward various L-amino acids. To investigate the role of Arg305 in substrate specificity, we constructed Arg305 variants of LGOX. In all mutants, the substrate specificity of LGOX was markedly changed by the mutation. The results of kinetics and pH dependence on activity indicate that Arg305 of LGOX is associated with the interaction of enzyme and side chain of substrate.

Structural characterization of L-glutamate oxidase from Streptomyces sp. X-119-6.

L-Glutamate oxidase (LGOX) from Streptomyces sp. X-119-6, which catalyzes the oxidative deamination of L-glutamate, has attracted increasing attention as a component of amperometric L-glutamate sensors used in the food industry and clinical biochemistry. The precursor of LGOX, which has a homodimeric structure, is less active than the mature enzyme with an alpha(2)beta(2)V(2) structure; enzymatic proteolysis of the precursor forms the stable mature enzyme. We solved the crystal structure of mature LGOX using molecular replacement with a structurally homologous model of L-amino acid oxidase (LAAO) from snake venom: LGOX has a deeply buried active site and two entrances from the surface of the protein into the active site. Comparison of the LGOX structure with that of LAAO revealed that LGOX has three regions that are absent from the LAAO structure, one of which is involved in the formation of the entrance. Furthermore, the arrangement of the residues composing the active site differs between LGOX and LAAO, and the active site of LGOX is narrower than that of LAAO. Results of the comparative analyses described herein raise the possibility that such a unique structure of LGOX is associated with its substrate specificity.

Determination of the activities of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase in a microfluidic system.

A microfluidic system for the analysis of the activities of glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) was fabricated. The device consists of a glass chip with a micro-electrochemical L-glutamate sensor and a polydimethylsiloxane (PDMS) sheet with a Y-shaped micro-flow channel. A sample solution and a substrate solution for the enzymes were introduced from two injection ports at the end of the flow channel. When the flows were stopped, substrates in a solution mixed immediately with either of the enzymes by diffusion in a mixing channel. L-glutamate produced by the enzymatic reaction of GOT or GPT in the flow channel was detected by using the L-glutamate sensor. A distinct current increase was observed immediately after mixing, and the initial slope of the response curve varied in proportion to the activity of GOT or GPT. The relation between the slope of the response curve and the enzyme activity was linear between 7 and 228 U l-1 for GOT and 9 and 250 U l-1 for GPT. The quality of the response curve was improved with an increase in the channel height. The measurement based on the rate analysis in the micro-flow channel facilitated the reduction of the influence of interferents. The influence of the viscosity of the sample solution was also checked for the analysis of real samples. The determination of the enzyme activities was also conducted in a system with micropumps fabricated for a sample injection. Two solutions could be mixed in the mixing channel, and the activity of the enzymes could be measured as in the experiments using microsyringe pumps.

Electrochemical determination of gamma-glutamyl transpeptidase activity and its application to a miniaturized analysis system.

A novel method to determine the activity of gamma-glutamyl transpeptidase (gamma-GTP) was developed. gamma-l-glutamyl-l-glutamate and glycyl-glycine were used as the substrates for gamma-GTP. l-glutamate produced by the enzymatic reaction was measured with an amperometric l-glutamate sensor. Following the mixing of the substrate solution and a sample solution, the current generated on the l-glutamate sensor continued to increase at a constant rate. The method was used to construct a miniaturized analysis system for the determination of gamma-GTP activity. The system consisted of the l-glutamate sensor formed on a glass substrate and a polydimethylsiloxane (PDMS) flow channel. Since the l-glutamate concentration in the solution increased as the solution was mobilized through the flow channel, a constant current increase was observed. The relation between the slope of the response curve and the activity of gamma-GTP was linear between 35 U l(-1) and 659 U l(-1). The rate analysis in the micro flow channel minimized the influence of interferents. The reproducibility of the output of the micro system was found to be good with a relative standard deviation (R.S.D.) of 5.6% at 659 U l(-1). The activities of gamma-GTP in human serum samples were also determined and compared with values obtained with a conventional spectroscopic method. The values obtained by the two methods were consistent with a correlation coefficient of 0.953.

Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6.

L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.