L-amino Acid Oxidase Research Articles

TMET-15. INVESTIGATING THE IL4I1-AHR AXIS IN GLIOBLASTOMA METABOLISM

Abstract Glioblastoma (GB) is the most frequent and malignant form of primary brain tumors in adults. Despite multi-modal therapy consisting of surgery, radio- and chemotherapy, tumors recur and overall survival remains poor. Previously, we identified that besides the classical tryptophan-catabolizing enzyme tryptophan-2,3-dioxygenase (TDO2), the L-amino acid oxidase interleukin-4-induced 1 (IL4I1) is able to activate the transcription factor aryl hydrocarbon receptor (AHR) via production of downstream metabolites. Activation of the AHR in GB may promote tumor progression e.g. by inhibiting anti-tumor immune responses. In this study, we aim to elucidate further the specific effects of the metabolic enzyme IL4I1 and its metabolites on AHR activation in GB. Further, we set out to identify and validate novel AHR target genes downstream of IL4I1. Harnessing the IL4I1-AHR axis might lead to the development of novel therapy strategies helping to overcome resistance in GB in the future.

L-Amino Acid Oxidase from Marine Bacterium: Purification, Characterization and Evaluation of its Anticancer and antioxidant activities

L-Amino Acid Oxidase from Marine Bacterium: Purification, Characterization and Evaluation of its Anticancer and antioxidant activities

The Toxin Diversity, Cytotoxicity, and Enzymatic Activity of Cape Cobra (Naja nivea) Venom.

"True" cobras (genus Naja) are among the venomous snakes most frequently involved in snakebite accidents in Africa and Asia. The Cape cobra (Naja nivea) is one of the African cobras of highest medical importance, but much remains to be learned about its venom. Here, we used a shotgun proteomics approach to better understand the qualitative composition of N. nivea venom and tested its cytotoxicity and protease activity as well as its effect on intracellular Ca2+ release and NO synthesis. We identified 156 venom components representing 17 protein families, with the dominant ones being three-finger toxins, mostly of the short-chain type. Two-thirds of the three-finger toxin entries identified were assigned as cytotoxins, while the remainder were categorized as neurotoxins, including short-chain, long-chain, and ancestral three-finger toxins. We also identified snake venom metalloproteinases and members of CRISP, l-amino acid oxidase, and other families. Protease activity and its effect on intracellular Ca2+ release and NO synthesis were low. Phospholipase A2 activity was surprisingly high, despite this toxin family being marginally recovered in the analyzed venom. Cytotoxicity was relevant only at higher venom concentrations, with macrophage and neuroblastoma cell lines showing the lowest viability. These results are in line with the predominantly neurotoxic envenomation symptoms caused by Cape cobra bites. The present overview of the qualitatively complex and functionally intriguing venom of N. nivea may provide insights into the pathobiochemistry of this species' venom.

Mesenchymal stromal cells restrain the Th17 cell response via L-amino-acid oxidase within lymph nodes

Mesenchymal stromal/stem cells (MSC) have emerged as a promising therapeutic avenue for treating autoimmune diseases, eliciting considerable interest and discussion regarding their underlying mechanisms. This study revealed the distinctive ability of human umbilical cord MSC to aggregate within the lymph nodes of mice afflicted with autoimmune diseases, but this phenomenon was not observed in healthy mice. The specific distribution is driven by the heightened expression of the CCL21-CCR7 axis in mice with autoimmune diseases, facilitating the targeted homing of MSC to the lymph nodes. Within the lymph nodes, MSC exhibit a remarkable capacity to modulate Th17 cell function, exerting a pronounced anti-inflammatory effect. Transplanted MSC stimulates the secretion of L-amino-acid oxidase (LAAO), a response triggered by elevated levels of tumor necrosis factor-α (TNF-α) in mice with autoimmune diseases through the NF-κB pathway. The presence of LAAO is indispensable for the efficacy of MSC, as it significantly contributes to the inhibition of Th17 cells. Furthermore, LAAO-derived indole-3-pyruvic acid (I3P) serves as a potent suppressor of Th17 cells by activating the aryl hydrocarbon receptor (AHR) pathway. These findings advance our understanding of the global immunomodulatory effects exerted by MSC, providing valuable information for optimizing therapeutic outcomes.

Identification of antigenic proteins from the venom of Malaysian snakes using immunoprecipitation assay and tandem mass spectrometry (LC-MS/MS)

Snake envenomation poses a significant risk to Malaysians and country visitors. Malaysia witnesses an estimated 650 snake bites per 100,000 population annually. The primary treatment for snake envenomation involves administering antivenom derived from horses, despite its drawbacks, such as anaphylactic reactions and serum sickness. Identifying the venom proteome is crucial for understanding and predicting the clinical implications of envenomation and developing effective treatments targeting specific venom proteins. In this study, we employ an immunoprecipitation assay followed by LC-MS/MS to identify antigenic proteins in five common venomous snakes in Malaysia compassing of two families which are pit vipers, (Calloselasma rhodostoma and Cryptelytrops purpureomaculatus) and cobras (Ophiophagus hannah, Naja kaouthia, and Naja sumatrana). The immunoprecipitation assay utilises a 2 % agarose gel, allowing antigenic proteins to diffuse and bind with antibodies in the antivenom. The antivenom utilised in this research was procured from the Queen Saovabha Memorial Institute (QSMI), Thailand, including king cobra antivenom (KCAV), cobra antivenom (CAV), Malayan pit viper antivenom (MPAV), Russell's viper antivenom (RPAV), hematopolyvalent antivenom (HPAV), neuropolyvalent antivenom (NPAV), banded krait antivenom (BKAV), and Malayan krait antivenom (MKAV). The protein identified through these interactions which are exclusive to the cobras are three-finger toxins (3FTXs) while snake C-type lectins (Snaclecs) are unique to the pit vipers. Common protein that are present in both families are L-amino acid oxidase (LAAO), Phospholipase A2 (PLA2), and snake venom metalloproteinase (SVMP). Identifying these proteins is vital for formulating a broad-spectrum antivenom applicable across multiple species.

Crystal structure and enzyme engineering of the broad substrate spectrum l-amino acid oxidase 4 from the fungus Hebeloma cylindrosporum.

l-Amino acid oxidases (LAAOs) catalyze the oxidative deamination of l-amino acids to α-keto acids. Recombinant production of LAAOs with broad substrate spectrum remains a formidable challenge. We previously achieved this for the highly active and thermostable LAAO4 of Hebeloma cylindrosporum (HcLAAO4). Here, we crystallized a proteolytically truncated surface entropy reduction variant of HcLAAO4 and solved its structure in substrate-free form and in complex with diverse substrates. The ability to support the aliphatic portion of a substrate's side chain by an overall hydrophobic active site is responsible for the broad substrate spectrum of HcLAAO4, including l-amino acids with big aromatic, acidic and basic side chains. Based on the structural findings, we generated an E288H variant with increased activity toward pharmaceutical building blocks of high interest.

Interfacially crystallized Enzyme@MOFs biocatalytic membranes for highly efficient synthesis of D-amino acids via continuously recirculating stereoinversion

D-Amino acids (D-AAs) constitute essential building blocks found in many pharmaceuticals and fine chemicals. We herein presented the coating of polyvinylidene fluoride (PVDF) hollow fiber membrane with L-amino acid oxidase (LAAO) embedded zeolitic imidazolate framework-8 (ZIF-8) layer by a single-step interfacial biomineralization strategy for efficient synthesis of D-AAs. The ZIF-8 skeleton and PVDF membrane structure not only guaranteed free enzyme activity and stability, but also reinforced the applicability of this bioreactor for continuous enzymatic reactions. By placing the prepared biocatalytic membrane in the constructed circulation device, only the L-AAs can be specifically oxidized. The D-AAs are accumulated and synthesized after multiple rounds of continuously recirculating stereoinversion. Impressively, using tryptophan (Trp) as the model molecule, the thus-designed bioreactor exhibited an excellent conversion rate (92.10%) and ee% (99.41%) of D-Trp with a shorter time under constant flow conditions. This study provides an unprecedented protocol for highly efficient synthesis of D-AAs, and immensely advances the promising application field of membrane bioreactors.

Biotechnological potential of Brazilian snake venom in the production of biologically active drugs: Literature review

Despite being feared, Brazilian venomous snakes can be reservoirs of important resources of medicinal interest, since the venoms of these animals have an invaluable potential for biologically active substances found in nature. Pharmacological and biochemical studies carried out in recent decades have shown the diversity of proteins with enzymatic activity, toxins, peptides, bioactive amines, among other compounds in snake venoms. The aim of this literature review was to describe the pharmaceutical biotechnological potential of Brazilian snake venom and to demonstrate the importance of these toxins and their pharmacological derivatives for public health. Since 1894, when they were studied by Vital Brazil, molecules derived from Brazilian snake venom have shown significant biological activity. These molecules have been proven to be efficient antimicrobial, antifungal and antiprotozoal agents, mainly due to their activity as serine proteases, hyaluronidases, L-amino acid oxidases (LAAO) and acetylcholinesterases, among other well-known substances. Brazilian snake venoms have thus been the target of a series of studies, which have borne excellent fruit, for example in the development of various drugs, such as Captopril®, which is derived from a peptide isolated from the venom of Bothrops jararaca, and fibrin sealant, derived from molecules in the venom of Crotalus durissus terrificus associated with cryoprecipitate from the blood plasma of buffaloes. With the analysis of biotechnologies applied to various molecules with the capacity for pharmacological, biologically active action, it is clear that this field is vast for new research and due to the great diversity of compounds in snake venoms. Certainly, as research progresses, new biologically active drugs derived from snake venom should become available for use and benefit of the population. Thus, studies related to the chemical composition of snake venoms should be encouraged, as well as those related to their pharmaceutical biotechnological potential.

Venom variation among the three subspecies of the North African mountain viper Vipera monticola Saint Girons 1953

The North African mountain viper (Vipera monticola) is a medically relevant venomous snake distributed in Morocco, Algeria, and Tunisia. Three subspecies of V. monticola, exhibiting differences in morphotypes and dietary regimes, are currently recognised: V. m. monticola, V. m. atlantica, and V. m. saintgironsi. Through the application of snake venomics, we analysed the venoms of specimens of Moroccan origin belonging to each of the three subspecies. Snake venom metalloproteinases (svMP), snake venom serine proteases (svSP), C-type lectin and C-type lectin-related proteins (CTL), and phospholipases A2 (PLA2) were predominant, with PLA2 being the most abundant toxin family overall. Disintegrins (DI) and cysteine-rich secretory proteins (CRISP) were exclusive to V. m. monticola and V. m. atlantica, while l-amino-acid oxidases (LAAO) were only found in V. m. saintgironsi. The differences detected in the venom profiles, as well as in presence/absence and relative abundances of toxin families, indicate the occurrence of intraspecific venom variation within V. monticola. The identified patterns of venom similarity between subspecies seem to align more with their phylogenetic relationships than with the reported differences in their feeding habits.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. The results highlight the need for species-specific antivenom to better manage snakebite victims.

Improvement of Heterologous Soluble Expression of L-amino Acid Oxidase Using Logistic Regression.

Successful implementation of enzymes in practical application hinges on the development of efficient mass production techniques. However, in a heterologous expression system, the protein is often unable to fold correctly and, thus, forms inclusion bodies, resulting in the loss of its original activity. In this study, we present a new and more accurate model for predicting amino acids associated with an increased L-amino acid oxidase (LAO) solubility. Expressing LAO from Rhizoctonia solani in Escherichia coli and combining random mutagenesis and statistical logistic regression, we modified 108 amino acid residues by substituting hydrophobic amino acids with serine and hydrophilic amino acids with alanine. Our results indicated that specific mutations in Euclidean distance, glycine, methionine, and secondary structure increased LAO expression. Furthermore, repeated mutations were performed for LAO based on logistic regression models. The mutated LAO displayed a significantly increased solubility, with the 6-point and 58-point mutants showing a 2.64- and 4.22-fold increase, respectively, compared with WT-LAO. Ultimately, using recombinant LAO in the biotransformation of α-keto acids indicates its great potential as a biocatalyst in industrial production.

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.

Genomic insights into the cellular specialization of predation in raptorial protists

BackgroundPredation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited.ResultsIn this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and l-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria.ConclusionsOur results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.

Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 μmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 μg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 μg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Development of an Enzyme Cascade System for the Synthesis of Enantiomerically Pure D-Amino Acids Utilizing Ancestral L-Amino Acid Oxidase.

Enantiomerically pure D-amino acids hold significant potential as precursors for synthesizing various fine chemicals, including peptide-based drugs and other pharmaceuticals. This study focuses on establishing an enzymatic cascade system capable of converting various L-amino acids into their D-isomers. The system integrates four enzymes: ancestral L-amino acid oxidase (AncLAAO-N4), D-amino acid dehydrogenase (DAADH), D-glucose dehydrogenase (GDH), and catalase. AncLAAO-N4 initiates the process by converting L-amino acids to corresponding keto acids, which are then stereo-selectively aminated to D-amino acids by DAADH using NADPH and NH4Cl. Concurrently, any generated H2O2 is decomposed into O2 and H2O by catalase, while GDH regenerates NADPH from D-glucose. Optimization of reaction conditions and substrate concentrations enabled the successful synthesis of five D-amino acids, including a D-Phe derivative, three D-Trp derivatives, and D-phenylglycine, all with high enantiopurity (>99 % ee) at a preparative scale (>100 mg). This system demonstrates a versatile approach for producing a diverse array of D-amino acids.

Toxins profiles, toxicological properties, and histological alteration potentiality of Trimeresurus erythrurus venom: In vitro and in vivo experiments

ObjectivesTrimeresurus erythrurus (Red-tailed bamboo pit viper) contributes significantly to snakebite envenoming cases in Bangladesh, India, Myanmar, and Nepal. However, the absence of specific antivenom and challenging socio-economic conditions in these regions necessitate the development of symptom-based medication to mitigate the severity of T. erythrurus envenoming. Therefore, documenting the effects of T. erythrurus venom on mammalian organs can establish a foundation for further research on symptom-based medication. MethodsWe profiled T. erythrurus venom toxins using SDS-PAGE. Additionally, we assessed the venom's toxicological properties through lethality, hemorrhagic, edematogenic, hemolytic, and phospholytic assays. Furthermore, we examined the histological alterations induced by crude venom on various organs of mice. ResultsSDS-PAGE profiling confirmed the presence of Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs), Phospholipase A2 (PLA2), L-amino Acid Oxidases (LAAOs), Myotoxins, and Disintegrins as toxic components in T. erythrurus venom. The venom exhibited a Median Lethal Dose (LD50) of 1.131 mg/kg, Minimum Hemorrhagic Dose (MHD) of 0.21 mg/kg, Minimum Edematogenic Dose (MED) of 0.17 mg/kg, and Median Hemolytic Dose (HD50) of 2.527 mg/L. Moreover, the venom induced significant pathological changes in mammalian skin, skeletal muscle, lung, heart, kidney, and intestine. ConclusionsThis study elucidates the major toxins, critical toxicological indices, and histopathological effects of T. erythrurus venom on mammalian organs. These findings provide valuable insights on the development of effective treatment strategies for pathophysiological complications that could manifest post T. erythrurus envenoming.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 μg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Proteomic Analysis and Immunoprofiling of Persian Horned Viper Venom, Pseudocerastes Persicus, from Central Part of Iran.

Numerous species of venomous snakes of medical importance exist in Iran. Pseudocerastes persicus (P. persicus), one of the medically important snakes, also called the Persian horned viper, has a geographical spread that extends to the east, southwest, and central areas of Iran and is endemic across the wider region. As a result, this species is responsible for many snakebite occurrences. Venom from P. persicus found in the central province of Semnan contains phospholipase A2 and L-amino acid oxidase activities, and high toxic potency. The venom was fractionated by reverse-phase high-performance liquid chromatography (HPLC) and analyzed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and two-dimensional electrophoresis. Using liquid chromatography with tandem mass spectrometry (LC-MS/MS), a range of components were identified, consistent with the biochemical and toxicological properties of the venom. Proteins identified from 2D electrophoresis and shotgun methods included metallo- and serine proteases, phospholipases, oxidases, and Kunitz trypsin inhibitors, along with many other components at lower qualitative abundance. This study provides a more detailed understanding of the protein profile of Iranian P. persicus venom, which can be effective in the production of an effective antidote against it. The analysis of the resulting data shows that there is a wide range of proteins in the venom of the Persian horned viper. This information can provide a better understanding of how venom is neutralized by polyclonal antivenom. Considering the wide presence of this snake and its related species in Iran and surrounding countries, knowing the venom protein profile of this family can be of great support to antivenom producers such as Razi Vaccine & Serum Research Institute in the preparation of regional antivenoms.

Proteomic Investigation of Cape Cobra (Naja nivea) Venom Reveals First Evidence of Quaternary Protein Structures.

Naja nivea (N. nivea) is classed as a category one snake by the World Health Organization since its envenomation causes high levels of mortality and disability annually. Despite this, there has been little research into the venom composition of N. nivea, with only one full venom proteome published to date. Our current study separated N. nivea venom using size exclusion chromatography before utilizing a traditional bottom-up proteomics approach to unravel the composition of the venom proteome. As expected by its clinical presentation, N. nivea venom was found to consist mainly of neurotoxins, with three-finger toxins (3FTx), making up 76.01% of the total venom proteome. Additionally, cysteine-rich secretory proteins (CRISPs), vespryns (VESPs), cobra venom factors (CVFs), 5'-nucleotidases (5'NUCs), nerve growth factors (NGFs), phospholipase A2s (PLA2), acetylcholinesterases (AChEs), Kunitz-type serine protease inhibitor (KUN), phosphodiesterases (PDEs), L-amino acid oxidases (LAAOs), hydrolases (HYDs), snake venom metalloproteinases (SVMPs), and snake venom serine protease (SVSP) toxins were also identified in decreasing order of abundance. Interestingly, contrary to previous reports, we find PLA2 toxins in N. nivea venom. This highlights the importance of repeatedly profiling the venom of the same species to account for intra-species variation. Additionally, we report the first evidence of covalent protein complexes in N. nivea venom, which likely contribute to the potency of this venom.

Reprogramming the Transition States to Enhance C-N Cleavage Efficiency of Rhodococcus opacusl-Amino Acid Oxidase.

l-Amino acid oxidase (LAAO) is an important biocatalyst used for synthesizing α-keto acids. LAAO from Rhodococcus opacus (RoLAAO) has a broad substrate spectrum; however, its low total turnover number limits its industrial use. To overcome this, we aimed to employ crystal structure-guided density functional theory calculations and molecular dynamic simulations to investigate the catalytic mechanism. Two key steps were identified: S → [TS1] in step 1 and Int1 → [TS2] in step 2. We reprogrammed the transition states [TS1] and [TS2] to reduce the identified energy barrier and obtain a RoLAAO variant capable of catalyzing 19 kinds of l-amino acids to the corresponding high-value α-keto acids with a high total turnover number, yield (≥95.1 g/L), conversion rate (≥95%), and space-time yields ≥142.7 g/L/d in 12-24 h, in a 5 L reactor. Our results indicated the promising potential of the developed RoLAAO variant for use in the industrial production of α-keto acids while providing a potential catalytic-mechanism-guided protein design strategy to achieve the desired physical and catalytic properties of enzymes.