Amino Acid Oxidase Research Articles

PEGylated Recombinant Aplysia punctata Ink Toxin Depletes Arginine and Lysine and Inhibits the Growth of Tumor Xenografts.

In recent years, a novel treatment method for cancer has emerged, which is based on the starvation of tumors of amino acids like arginine. The deprivation of arginine in serum is based on enzymatic degradation and can be realized by arginine deaminases like the l-amino acid oxidase found in the ink toxin of the sea hare Aplysia punctata. Previously isolated from the ink, the l-amino acid oxidase was described to oxidate the essential amino acids l-lysine and l-arginine to their corresponding deaminated alpha-keto acids. Here, we present the recombinant production and functionalization of the amino acid oxidase Aplysia punctata ink toxin (APIT). PEGylated APIT (APIT-PEG) increased the blood circulation time. APIT-PEG treatment of patient-derived xenografted mice shows a significant dose-dependent reduction of tumor growth over time mediated by amino acid starvation of the tumor. Treatment of mice with APIT-PEG, which led to deprivation of arginine, was well tolerated.

Myeloid cell-associated aromatic amino acid metabolism facilitates CNS myelin regeneration

Regulation of myeloid cell activity is critical for successful myelin regeneration (remyelination) in demyelinating diseases, such as multiple sclerosis (MS). Here, we show aromatic alpha-keto acids (AKAs) generated from the amino acid oxidase, interleukin-4 induced 1 (IL4I1), promote efficient remyelination in mouse models of MS. During remyelination, myeloid cells upregulated the expression of IL4I1. Conditionally knocking out IL4I1 in myeloid cells impaired remyelination efficiency. Mice lacking IL4I1 expression exhibited a reduction in the AKAs, phenylpyruvate, indole-3-pyruvate, and 4-hydroxyphenylpyruvate, in remyelinating lesions. Decreased AKA levels were also observed in people with MS, particularly in the progressive phase when remyelination is impaired. Oral administration of AKAs modulated myeloid cell-associated inflammation, promoted oligodendrocyte maturation, and enhanced remyelination in mice with focal demyelinated lesions. Transcriptomic analysis revealed AKA treatment induced a shift in metabolic pathways in myeloid cells and upregulated aryl hydrocarbon receptor activity in lesions. Our results suggest myeloid cell-associated aromatic amino acid metabolism via IL4I1 produces AKAs in demyelinated lesions to enable efficient remyelination. Increasing AKA levels or targeting related pathways may serve as a strategy to facilitate the regeneration of myelin in inflammatory demyelinating conditions.

Aerobic degradation of ketoprofen by marine consortia: Fenton-like reaction and degradation pathway

Ketoprofen (KTP) as a non-steroidal anti-inflammatory drug has been detected in coastal environment due to its wide usage. However, little information is available about the fate of KTP in marine environment. In the present study, the aerobic degradation of 20 mg L−1 KTP using the enriched marine consortia was investigated. Results showed that CA consortium cultured with casamino acids exhibited a higher KTP-degrading ability than those cultured with glucose, yeast extract and mixed vitamins. During CA consortium-mediated degradation of KTP, additional casamino acids resulted in the production of H2O2 and OH. Fe(III) could be also reduced to Fe(II) by CA consortium. This result indicated the occurrence of Fenton-like reaction. Further studies found that both biogenic Fenton-like reaction and enzyme-catalyzed reactions were involved in the initial hydroxylation reaction of KTP, then the subsequent mineralization of KTP was only performed via enzyme-catalyzed reactions. High-throughput sequencing analysis showed that Halomonas, Marinobacter, Owenweeksia and Oceanimonas were significantly enriched in CA consortium. As these genera contain amino acid oxidases, and the former two genera are capable of reducing Fe(III), it is assumed that these genera participated in biogenic Fenton-like reaction. The involvement of biogenic Fenton-like reaction provides a new insight into understanding the fate of KTP and other similar organic pollutants in marine environment containing amino acids and iron.

Computational Mechanistic Study of l-Aspartate Oxidase by ONIOM Method.

l-Aspartate oxidase (Laspo) is responsible for the oxidation of l-aspartate into iminoaspartate using flavin as a cofactor. During this process flavin is reduced, and it can be reoxidized by either molecular oxygen or fumarate. The overall fold and the catalytic residues of Laspo are similar to succinate dehydrogenase and fumarate reductase. On the basis of deuterium kinetic isotope effects as well as other kinetic and structural data, it is proposed that the enzyme can catalyze the oxidation of l-aspartate through a mechanism similar to amino acid oxidases. It is suggested that a proton is removed from the α-amino group, while a hydride is transferred from C2 to flavin. It is also suggested that the hydride transfer is a rate-limiting step. However, there is still an ambiguity about the stepwise or concerted mechanism of hydride- and proton-transfer steps. In this study, we formulated some computational models to study the hydride-transfer mechanism using the crystal structure of Escherichia colil-aspartate oxidase in complexes with succinate. The calculations involved our own N-layered integrated molecular orbital and molecular mechanics method, and we evaluated the geometry and energetics of the hydride/proton-transfer processes while probing the roles of active site residues. Based on the calculations, it is concluded that proton- and hydride-transfer steps are decoupled, and a stepwise mechanism might be operative as opposed to the concerted one.

The Symmetric Active Site of Enantiospecific Enzymes

Biomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry.

PS-B06-4: ADENINE NEPHROPATHY IS ALLEVIATED BY ANTI-INFLAMMATORY PROPERTY OF SODIUM BENZOATE

Background: D-amino acid oxidase (DAO), a molecule distributed in brain, liver, and kidney in human, oxidases d-amino acids to alpha-keto acids to produce hydrogen peroxides. Sodium benzoate (SB), a known DAO enzyme inhibitor, has anti-inflammatory property, although its effect has not been investigated in chronic kidney disease (CKD). Methods: Adenine induced CKD (AdCKD) or 2,8-dihydroxyadenine (2,8-DHA) nephropathy is applied for rodent CKD studies. AdCKD was induced by oral administration of adenine in C57BL/6JJcl mice with or without SB oral administration. AdCKD was also induced in DAO enzyme deficient mice (DAO-/-) and treated with or without SB. Human THP-1 cells and macrophages induced from THP-1 cells by phorbol 12-myristate 13-acetate were stimulated by LPS or TNF-alpha with or without SB to confirm the anti-inflammatory effect of SB. Results: AdCKD showed increased serum creatinine and urea nitrogen levels as well as kidney interstitial fibrosis and tubular atrophy score, which were significantly attenuated by the treatment of SB. Survival of AdCKD mice were significantly prolonged by 2.6-fold by SB administration. SB significantly decreased elevated kidney positive F4/80 immunohistochemistry area and renal mRNA level of macrophage markers and inflammatory markers of AdCKD. Human THP-1 cells and macrophages stimulated either by LPS or human TNF-alpha showed increased inflammatory gene mRNA expressions, which were significantly reduced by SB treatment. Conclusions: SB significantly ameliorated renal damage in AdCKD by suppressing inflammatory gene expressions and macrophage infiltration.

Recombinant l-Amino Acid Oxidase with Broad Substrate Spectrum for Co-substrate Recycling in (S)-Selective Transaminase-Catalyzed Kinetic Resolutions.

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co‐substrate. A dual‐enzyme recycling system overcomes this limitation: l‐amino acid oxidases (LAAO) recycle the accumulating co‐product of (S)‐selective transaminases in the kinetic resolution of racemic amines to produce pure (R)‐amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub‐stoichiometric amounts of 1 mol% of the transaminase co‐substrate as well as the initial application of l‐amino acids instead of α‐keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)‐methylbenzylamine (>99 %ee) at complete conversion (50 %).

Profilings of subproteomes of lectin-binding proteins of nine Bothrops venoms reveal variability driven by different glycan types

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

Monitoring and control of the release of soluble O2 from H2 O2 inside porous enzyme carrier for O2 supply to an immobilized d-amino acid oxidase.

While O2 substrate for bio‐transformations in bulk liquid is routinely provided from entrained air or O2 gas, tailored solutions of O2 supply are required when the bio‐catalysis happens spatially confined to the microstructure of a solid support. Release of soluble O2 from H2O2 by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O2 formed by an immobilized catalase upon feeding of H2O2. The internally released O2 is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H2O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100–500 µM. Thus, enzyme inactivation by excess H2O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air‐saturated level. Conversions at a 200 ml scale using varied H2O2 feed rate (0.04–0.18 mmol/min) give the equivalent production rate from d‐methionine (200 mM) and achieve rate enhancement by ∼1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O2 substrate from H2O2 into carrier‐immobilized enzymes. By addressing limitations of O2 supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2‐dependent biocatalytic reactions.

Simultaneous Coordination of Zn(II) and Zr(IV) to d-Amino Acid Oxidase: Highly Efficient Catalytic Transfer Hydrogenation of Levulinic Acid to γ-Valerolactone

Simultaneous Coordination of Zn(II) and Zr(IV) to <scp>d</scp>-Amino Acid Oxidase: Highly Efficient Catalytic Transfer Hydrogenation of Levulinic Acid to γ-Valerolactone

AutoDesigner, a De Novo Design Algorithm for Rapidly Exploring Large Chemical Space for Lead Optimization: Application to the Design and Synthesis of d-Amino Acid Oxidase Inhibitors.

The lead optimization stage of a drug discovery program generally involves the design, synthesis, and assaying of hundreds to thousands of compounds. The design phase is usually carried out via traditional medicinal chemistry approaches and/or structure-based drug design (SBDD) when suitable structural information is available. Two of the major limitations of this approach are (1) difficulty in rapidly designing potent molecules that adhere to myriad project criteria, or the multiparameter optimization (MPO) problem, and (2) the relatively small number of molecules explored compared to the vast size of chemical space. To address these limitations, we have developed AutoDesigner, a de novo design algorithm. AutoDesigner employs a cloud-native, multistage search algorithm to carry out successive rounds of chemical space exploration and filtering. Millions to billions of virtual molecules are explored and optimized while adhering to a customizable set of project criteria such as physicochemical properties and potency. Additionally, the algorithm only requires a single ligand with measurable affinity and a putative binding model as a starting point, making it amenable to the early stages of an SBDD project where limited data are available. To assess the effectiveness of AutoDesigner, we applied it to the design of novel inhibitors of d-amino acid oxidase (DAO), a target for the treatment of schizophrenia. AutoDesigner was able to generate and efficiently explore over 1 billion molecules to successfully address a variety of project goals. The compounds generated by AutoDesigner that were synthesized and assayed (1) simultaneously met not only physicochemical criteria, clearance, and central nervous system (CNS) penetration (Kp,uu) cutoffs but also potency thresholds and (2) fully utilize structural data to discover and explore novel interactions and a previously unexplored subpocket in the DAO active site. The reported data demonstrate that AutoDesigner can play a key role in accelerating the discovery of novel, potent chemical matter within the constraints of a given drug discovery lead optimization campaign.

A Review of Electrochemical Sensors for the Detection of Glycated Hemoglobin.

Glycated hemoglobin (HbA1c) is the gold standard for measuring glucose levels in the diagnosis of diabetes due to the excellent stability and reliability of this biomarker. HbA1c is a stable glycated protein formed by the reaction of glucose with hemoglobin (Hb) in red blood cells, which reflects average glucose levels over a period of two to three months without suffering from the disturbance of the outside environment. A number of simple, high-efficiency, and sensitive electrochemical sensors have been developed for the detection of HbA1c. This review aims to highlight current methods and trends in electrochemistry for HbA1c monitoring. The target analytes of electrochemical HbA1c sensors are usually HbA1c or fructosyl valine/fructosyl valine histidine (FV/FVH, the hydrolyzed product of HbA1c). When HbA1c is the target analyte, a sensor works to selectively bind to specific HbA1c regions and then determines the concentration of HbA1c through the quantitative transformation of weak electrical signals such as current, potential, and impedance. When FV/FVH is the target analyte, a sensor is used to indirectly determine HbA1c by detecting FV/FVH when it is hydrolyzed by fructosyl amino acid oxidase (FAO), fructosyl peptide oxidase (FPOX), or a molecularly imprinted catalyst (MIC). Then, a current proportional to the concentration of HbA1c can be produced. In this paper, we review a variety of representative electrochemical HbA1c sensors developed in recent years and elaborate on their operational principles, performance, and promising future clinical applications.

Pt Nanoparticles with Enhanced Deaminase‐like Activity: Example of Oxidative Deamination of 5‐Hydroxymethylfurfurylamine and Glutamic Acid

AbstractOxidative deamination is part of the metabolic pathways of amino acid oxidase and glutamate dehydrogenase enzymes, and enables obtaining α‐keto acid from amino compound with ammonia as a by‐product. This work presents the utilization of Pt‐based catalyst composed of small platinum nanoparticles supported on industrial silica support (Pt/SiO2). This nanozyme was tested in the oxidative deamination reaction of two amino compounds: 5‐hydroxymethylfurfurylamine (HMFA) to 5‐hydroxymethylfurfural (HMF) and glutamic acid (GA) to α‐ketoglutaric acid (KGA). This paper outlines the use of Pt nanozymes with deaminase‐like activity. We showed the ability of this nanozymes to be selective and active in liquid phase oxidative deamination of HMFA and glutamic acid in a 6 to 9 pH range at 60 °C under atmospheric pressure. Pt/SiO2 nanozyme actually plays as a deaminase mimic with high HMFA conversion (60%) and cumulative yield to the corresponding desaminated keto compounds (67%).

Male Infertility and Oxidative Stress: A Focus on the Underlying Mechanisms.

Reactive oxygen species (ROS) play a critical role in defining the functional competence of human spermatozoa. When generated in moderate amounts, ROS promote sperm capacitation by facilitating cholesterol efflux from the plasma membrane, enhancing cAMP generation, inducing cytoplasmic alkalinization, increasing intracellular calcium levels, and stimulating the protein phosphorylation events that drive the attainment of a capacitated state. However, when ROS generation is excessive and/or the antioxidant defences of the reproductive system are compromised, a state of oxidative stress may be induced that disrupts the fertilizing capacity of the spermatozoa and the structural integrity of their DNA. This article focusses on the sources of ROS within this system and examines the circumstances under which the adequacy of antioxidant protection might become a limiting factor. Seminal leukocyte contamination can contribute to oxidative stress in the ejaculate while, in the germ line, the dysregulation of electron transport in the sperm mitochondria, elevated NADPH oxidase activity, or the excessive stimulation of amino acid oxidase action are all potential contributors to oxidative stress. A knowledge of the mechanisms responsible for creating such stress within the human ejaculate is essential in order to develop better antioxidant strategies that avoid the unintentional creation of its reductive counterpart.

Reconstruction of Hyper‐Thermostable Ancestral L‐Amino Acid Oxidase to Perform Deracemization to D‐Amino Acids

AbstractL‐amino acid oxidases (LAAOs) with broad substrate specificity can be used in the deracemization of D,L‐amino acids (D,L‐AAs) to their D‐enantiomers. Hyper‐thermostable LAAO (HTAncLAAO) was designed through a combination of manual sequence data mining and ancestral sequence reconstruction. Soluble expression of HTAncLAAO (&gt;50 mg/L) can be achieved using an E. coli system. HTAncLAAO, which recognizes seven L‐AAs as substrates, exhibits extremely high thermal stability and long‐term stability; the t1/2 value was 95 °C and &lt;5% activity loss after incubation of the enzyme at 30 °C for 1 week. Deracemization could be achieved at 40 °C with a small amount of enzymes compared to previously reported LAAOs. A total of 0.4 mg (2 U) of HTAncLAAO is enough to deracemize three D,L‐AAs at a preparative scale with high enantiomeric excess (&gt;99 % ee, D‐enantiomer). These results suggest that HTAncLAAO is an excellent biocatalyst to perform this deracemization.

Analysis of N-glycosylation in fungal l-amino acid oxidases expressed in the methylotrophic yeast Pichia pastoris.

l‐amino acid oxidases (LAAOs) catalyze the oxidative deamination of l‐amino acids to corresponding α‐keto acids. Here, we describe the heterologous expression of four fungal LAAOs in Pichia pastoris. cgLAAO1 from Colletotrichum gloeosporioides and ncLAAO1 from Neurospora crassa were able to convert substrates not recognized by recombinant 9His‐hcLAAO4 from the fungus Hebeloma cylindrosporum described earlier thereby broadening the substrate spectrum for potential applications. 9His‐frLAAO1 from Fibroporia radiculosa and 9His‐laLAAO2 from Laccaria amethystine were obtained only in low amounts. All four enzymes were N‐glycosylated. We generated mutants of 9His‐hcLAAO4 lacking N‐glycosylation sites to further understand the effects of N‐glycosylation. All four predicted N‐glycosylation sites were glycosylated in 9His‐hcLAAO4 expressed in P. pastoris. Enzymatic activity was similar for fully glycosylated 9His‐hcLAAO4 and variants without one or all N‐glycosylation sites after acid activation of all samples. However, activity without acid treatment was low in a variant without N‐glycans. This was caused by the absence of a hypermannosylated N‐glycan on asparagine residue N54. The lack of one or all of the other N‐glycans was without effect. Our results demonstrate that adoption of a more active conformation requires a specific N‐glycosylation during biosynthesis.

IL4I1 Is Expressed by Head-Neck Cancer-Derived Mesenchymal Stromal Cells and Contributes to Suppress T Cell Proliferation.

Amino acids have a primary role in cancer metabolism. Beyond their primary biosynthetic role, they represent also an alternative fuel while their catabolites can influence the epigenetic control of gene expression and suppress anti-tumor immune responses. The accumulation of amino-acid derivatives in the tumor microenvironment depends not only on the activity of tumor cells, but also on stromal cells. In this study, we show that mesenchymal stromal cells derived from head–neck cancer express the amino acid oxidase IL4I1 that has been detected in different types of tumor cells. The catabolic products of IL4I1, H2O2, and kynurenines are known to suppress T cell response. We found that neutralization of IL4I1 activity can restore T cell proliferation. Thus, therapeutical strategies targeting enzymes involved in amino-acid catabolism may be helpful to contemporary block tumor cell migration and restore an efficacious anti-tumor immunity.

Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli

Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies.&#x0D; Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD).&#x0D; Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine.&#x0D; Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

Using D- and L-Amino Acid Oxidases to Generate the Imino Acid Substrate to Measure the Activity of the Novel Rid (Enamine/Imine Deaminase) Class of Enzymes.

This chapter describes a method to assay the activity of reactive intermediate deaminases (Rid), a large family of conserved soluble enzymes, which have been proposed to prevent damages from metabolic intermediates such as the highly reactive and unstable compounds enamines/imines. In this method, the flavin adenine dinucleotide-dependent L- or D-amino acid oxidases generate an imino acid starting from a L- or D- amino acid, respectively. This reaction is coupled to the hydrolysis of the imino acid to the corresponding α-keto acid and ammonium ion catalyzed by a Rid enzyme. The spectrophotometric assay consists of measuring the decrease of the initial rate of formation of the semicarbazone, derived from the spontaneous reaction of the imino acid and semicarbazide, caused by the presence of the Rid enzyme. The set-up and testing of this method imply a preliminary characterization of the ability of the amino acid oxidase to release the imino acid required for the subsequent reactions. To this purpose, the activity of the L- or D-amino acid oxidases with different amino acids can be measured as production of hydrogen peroxide or formation of semicarbazone in parallel assays. The advantages and limitations of this assay of Rid activity are discussed.