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• W3035559913 abstract "l-Lysine oxidase/monooxygenase (l-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of l-amino acids, particularly l-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (H2O2), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of l-lysine and l-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from l-lysine), l-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from l-lysine and 4-aminobutanoic acid from l-ornithine). The reaction of reduced enzyme and oxygen generated an imino acid and H2O2, with no detectable C4a-hydroperoxyflavin. Single-turnover reactions in which l-LOX/MOG was first reduced by l-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H2O2 addition after enzyme denaturation. H2O2 addition to active l-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that H2O2 generated from l-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H2O2 resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on l-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation. l-Lysine oxidase/monooxygenase (l-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of l-amino acids, particularly l-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (H2O2), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of l-lysine and l-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from l-lysine), l-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from l-lysine and 4-aminobutanoic acid from l-ornithine). The reaction of reduced enzyme and oxygen generated an imino acid and H2O2, with no detectable C4a-hydroperoxyflavin. Single-turnover reactions in which l-LOX/MOG was first reduced by l-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H2O2 addition after enzyme denaturation. H2O2 addition to active l-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that H2O2 generated from l-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H2O2 resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on l-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation. Enzymes are indispensable for living cells, as they are required to catalyze the reactions that serve physiological needs. Most enzymes catalyze a single specific reaction with the ability to use a certain set of compounds as substrates that are compatible with the binding and catalytic machinery. However, enzymes that employ a single active site to catalyze two types of reactions also exist. Their origin and the principles underlying their ability to accommodate dual activities are currently not well-understood. Nevertheless, the existence of these enzymes in nature is becoming more recognized. Understanding the general principles dictating their activities is useful for enzyme engineering or protein evolution to devise biocatalysts relevant for future industrial and biotechnology applications (1Copley S.D. Enzymes with extra talents: moonlighting functions and catalytic promiscuity.Curr. Opin. Chem. Biol. 2003; 7 (12714060): 265-27210.1016/S1367-5931(03)00032-2Crossref PubMed Scopus (426) Google Scholar, 2Khersonsky O. Roodveldt C. Tawfik D.S. Enzyme promiscuity: evolutionary and mechanistic aspects.Curr. Opin. Chem. Biol. 2006; 10 (16939713): 498-50810.1016/j.cbpa.2006.08.011Crossref PubMed Scopus (490) Google Scholar, 3Copley S.D. An evolutionary biochemist's perspective on promiscuity.Trends Biochem. Sci. 2015; 40 (25573004): 72-7810.1016/j.tibs.2014.12.004Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 4Copley S.D. Shining a light on enzyme promiscuity.Curr. Opin. Struct. Biol. 2017; 47 (29169066): 167-17510.1016/j.sbi.2017.11.001Crossref PubMed Scopus (93) Google Scholar). The characteristics of enzymes with dual activities in a single active site are diverse and can be found in a wide range of enzyme classes. Tetrachlorohydroquinone dehalogenase from Sphingomonas chlorophenolica catalyzes dehalogenation and isomerization of maleylactone and its analogues within a single active site (5Anandarajah K. Kiefer Jr., P.M. Donohoe B.S. Copley S.D. Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol.Biochemistry. 2000; 39 (10820000): 5303-531110.1021/bi9923813Crossref PubMed Scopus (95) Google Scholar). Acetohydroxyacid synthase (AHAS) from Thermotoga maritime catalyzes dual reactions with a major activity (∼90%) of converting pyruvate to acetoacetate and a minor (10%) pyruvate decarboxylase (PDC) activity that can convert pyruvate to acetaldehyde. Understanding how the PDC activity in AHAS is controlled is useful for the biofuel industry (6Eram M.S. Ma K. Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima.Biochem. Biophys. Rep. 2016; 7 (28955930): 394-39910.1016/j.bbrep.2016.07.008PubMed Google Scholar). Dual activities in a single active site have also been recognized in many flavoenzyme oxidases. However, insights into the mechanisms of these flavoenzymes and the factors dictating their activities are still lacking. Flavoenzyme oxidases generally catalyze oxidation of substrates with reduction of O2 to generate H2O2. Their reactions are useful for a wide range of applications, such as the production of valuable chemicals (7Dijkman W.P. de Gonzalo G. Mattevi A. Fraaije M.W. Flavoprotein oxidases: classification and applications.Appl. Microbiol. Biotechnol. 2013; 97 (23640366): 5177-518810.1007/s00253-013-4925-7Crossref PubMed Scopus (96) Google Scholar). Amino acid oxidases comprise a large class of flavin-dependent oxidases with two structural families, including monoamine oxidase (MAO) and d-amino acid oxidase (d-AAO) families (8Fitzpatrick P.F. Oxidation of amines by flavoproteins.Arch. Biochem. Biophys. 2010; 493 (19651103): 13-2510.1016/j.abb.2009.07.019Crossref PubMed Scopus (171) Google Scholar). Despite catalyzing the same conversion of amino acids to keto acids and H2O2, d-amino acid oxidases and l-amino acid oxidases (l-AAO) are classified into different structural families, with d-AAO in the d-AAO structural family and l-AAO in the MAO family (7Dijkman W.P. de Gonzalo G. Mattevi A. Fraaije M.W. Flavoprotein oxidases: classification and applications.Appl. Microbiol. Biotechnol. 2013; 97 (23640366): 5177-518810.1007/s00253-013-4925-7Crossref PubMed Scopus (96) Google Scholar). Enzymes in both d-AAO and l-AAO have been developed and applied as biosensors for detection of food contamination in nutritional industries (9Civitelli R. Villareal D.T. Agnusdei D. Nardi P. Avioli L.V. Gennari C. Dietary l-lysine and calcium metabolism in humans.Nutrition. 1992; 8 (1486246): 400-405PubMed Google Scholar, 10Mora M.F. Giacomelli C.E. Garcia C.D. Interaction of d-amino acid oxidase with carbon nanotubes: implications in the design of biosensors.Anal. Chem. 2009; 81 (19132842): 1016-102210.1021/ac802068nCrossref PubMed Scopus (52) Google Scholar), as indicators for metabolic and neurological disorders (11Turpin F. Dallerac G. Mothet J.P. Electrophysiological analysis of the modulation of NMDA-receptors function by d-serine and glycine in the central nervous system.Methods Mol. Biol. 2012; 794 (21956572): 299-31210.1007/978-1-61779-331-8_20Crossref PubMed Scopus (13) Google Scholar), and as biocatalysts in pharmaceutical industries (12Pollegioni L. Molla G. New biotech applications from evolved d-amino acid oxidases.Trends. Biotechnol. 2011; 29 (21397351): 276-28310.1016/j.tibtech.2011.01.010Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Füller J.J. Röpke R. Krausze J. Rennhack K.E. Daniel N.P. Blankenfeldt W. Schulz S. Jahn D. Moser J. Biosynthesis of violacein, structure and function of l-tryptophan oxidase VioA from Chromobacterium violaceum.J. Biol. Chem. 2016; 291 (27466367): 20068-2008410.1074/jbc.M116.741561Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). For example, l-lysine α-oxidase from Trichoderma viride (14Kelly S.C. O'Connell P.J. O'Sullivan C.K. Guilbault G.G. Development of an interferent free amperometric biosensor for determination of l-lysine in food.Anal. Chim. Acta. 2000; 412: 111-11910.1016/S0003-2670(00)00767-4Crossref Scopus (55) Google Scholar, 15Olschewski H. Erlenkötter A. Zaborosch C. Chemnitius G.C. 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Toxicol. 2014; 114 (24118879): 336-34310.1111/bcpt.12155Crossref PubMed Scopus (40) Google Scholar) and antimicrobial (22Yang H. Johnson P.M. Ko K.C. Kamio M. Germann M.W. Derby C.D. Tai P.C. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing l-amino acid oxidase from ink of the sea hare Aplysia californica.J. Exp. Biol. 2005; 208 (16155232): 3609-362210.1242/jeb.01795Crossref PubMed Scopus (100) Google Scholar) activities, suggesting their roles as protein drugs. Many l-AAOs, such as l-phenylalanine oxidase (PAO) from Pseudomonas sp. P-501 (23Ida K. Suguro M. Suzuki H. High resolution X-ray crystal structures of l-phenylalanine oxidase (deaminating and decarboxylating) from Pseudomonas sp. P-501. Structures of the enzyme-ligand complex and catalytic mechanism.J. Biochem. 2011; 150 (21841183): 659-66910.1093/jb/mvr103Crossref PubMed Scopus (15) Google Scholar), tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi (24Emanuele J.J. Fitzpatrick P.F. Mechanistic studies of the flavoprotein tryptophan 2-monooxygenase. 2. pH and kinetic isotope effects.Biochemistry. 1995; 34 (7893668): 3716-372310.1021/bi00011a029Crossref PubMed Scopus (36) Google Scholar, 25Sobrado P. Fitzpatrick P.F. Analysis of the role of the active site residue Arg98 in the flavoprotein tryptophan 2-monooxygenase, a member of the l-amino oxidase family.Biochemistry. 2003; 42 (14636049): 13826-1383210.1021/bi035299nCrossref PubMed Scopus (23) Google Scholar), and l-lysine monooxygenase (LMO) from Pseudomonas sp. (26Flashner M.I. Massey V. Purification and properties of l-lysine monooxygenase from Pseudomonas fluorescens.J. Biol. Chem. 1974; 249 (4207122): 2579-2586Abstract Full Text PDF PubMed Google Scholar), are known to contain two activities, oxidases and monooxygenases, in the same active sites. The monooxygenase is an apparent activity resulting from decarboxylation of an imino acid to form an amide product (Scheme 1). For some enzymes, such as LMO and TMO, which have structural characteristics classifying them as l-AAOs, their native major activity is monooxygenation of l-amino acid substrates (decarboxylation of imino acids) (Scheme 1) (26Flashner M.I. Massey V. Purification and properties of l-lysine monooxygenase from Pseudomonas fluorescens.J. Biol. Chem. 1974; 249 (4207122): 2579-2586Abstract Full Text PDF PubMed Google Scholar, 27Takeda H. Yamamoto S. Kojima Y. Hayaishi O. Studies on monooxygenases. I. General properties of crystalline l-lysine monooxygenase.J. Biol. Chem. 1969; 244 (5772467): 2935-2941Abstract Full Text PDF PubMed Google Scholar, 28Nakazawa T. Hori K. Hayaishi O. Studies on monooxygenases. V. Manifestation of amino acid oxidase activity by l-lysine monooxygenase.J. Biol. Chem. 1972; 247 (4624115): 3439-3444Abstract Full Text PDF PubMed Google Scholar, 29Yamamoto S. Yamauchi T. Hayaishi O. Alkylamine-dependent amino-acid oxidation by lysine monooxygenase–fragmented substrate of oxygenase.Proc. Natl. Acad. Sci. U. S. A. 1972; 69 (4509334): 3723-372610.1073/pnas.69.12.3723Crossref PubMed Scopus (8) Google Scholar). It should be noted that the monooxygenation activities of these l-AAO enzymes are different from true flavin-dependent monooxygenases, such as various phenolic monooxygenases (30Joosten V. van Berkel W.J. Flavoenzymes.Curr. Opin. Chem. Biol. 2007; 11 (17275397): 195-20210.1016/j.cbpa.2007.01.010Crossref PubMed Scopus (214) Google Scholar, 31Thotsaporn K. Chenprakhon P. Sucharitakul J. Mattevi A. Chaiyen P. Stabilization of C4a-hydroperoxyflavin in a two-component flavin-dependent monooxygenase is achieved through interactions at flavin N5 and C4a atoms.J. Biol. Chem. 2011; 286 (21680741): 28170-2818010.1074/jbc.M111.241836Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37 (22819837): 373-38010.1016/j.tibs.2012.06.005Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) or dechlorinating monooxygenase (33Pimviriyakul P. Thotsaporn K. Sucharitakul J. Chaiyen P. Kinetic mechanism of the dechlorinating flavin-dependent monooxygenase HadA.J. Biol. Chem. 2017; 292 (28159841): 4818-483210.1074/jbc.M116.774448Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 34Pimviriyakul P. Chaiyen P. A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin-dependent enzymes.J. Biol. Chem. 2018; 293 (30282807): 18525-1853910.1074/jbc.RA118.005538Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), in which one atom of a molecular O2 is truly incorporated into the substrates via formation of a reactive C4a-(hydro)peroxyflavin (32Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37 (22819837): 373-38010.1016/j.tibs.2012.06.005Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 35Huijbers M.M. Montersino S. Westphal A.H. Tischler D. van Berkel W.J. Flavin dependent monooxygenases.Arch. Biochem. Biophys. 2014; 544 (24361254): 2-1710.1016/j.abb.2013.12.005Crossref PubMed Scopus (334) Google Scholar, 36Romero E. Gómez Castellanos J.R. Gadda G. Fraaije M.W. Mattevi A. Same substrate, many reactions: oxygen activation in flavoenzymes.Chem. Rev. 2018; 118 (29323892): 1742-176910.1021/acs.chemrev.7b00650Crossref PubMed Scopus (197) Google Scholar). Although the reactions of flavin-dependent oxidases also involve an initial step of oxygen activation, most oxidases cannot facilitate the formation of a kinetically stable C4a-(hydro)peroxyflavin. The only exception is found in the reaction of pyranose 2-oxidase, which belongs to the class of glucose-methanol-choline oxidases in which C4a-hydroperoxyflavin first forms before H2O2 elimination takes place (37Chuaboon L. Wongnate T. Punthong P. Kiattisewee C. Lawan N. Hsu C.Y. Lin C.H. Bornscheuer U.T. Chaiyen P. One-pot bioconversion of l-arabinose to l-ribulose in an enzymatic cascade.Angew. Chem. 2019; 58 (30605256): 2428-243210.1002/anie.201814219Crossref Scopus (24) Google Scholar). For l-AAOs mentioned above, which can catalyze decarboxylation in addition to oxidation, their ability to form the C4a-hydroperoxyflavin intermediate is not clear. l-Amino acid oxidase/monooxygenase (l-AAO/MOG) from Pseudomonas sp. AIU 813 is an FAD-dependent oxidase that displays dual activities of oxidase and monooxygenase. l-Lysine is the most preferred substrate and is considered a native substrate for this enzyme. l-AAO/MOG showed significant oxidase activity (19%) and it is a different enzyme from the previously explored LMO, which displayed only monooxygenation activity (27Takeda H. Yamamoto S. Kojima Y. Hayaishi O. Studies on monooxygenases. I. General properties of crystalline l-lysine monooxygenase.J. Biol. Chem. 1969; 244 (5772467): 2935-2941Abstract Full Text PDF PubMed Google Scholar). To avoid confusion, in this report, we will refer to l-AAO/MOG from Pseudomonas sp. AIU 813 as l-lysine oxidase/monooxygenase (l-LOX/MOG). l-LOX/MOG is an ideal system for investigating the mechanism controlling the dual activities of a flavoenzyme oxidase. l-LOX/MOG catalyzes the minority reaction of oxidative deamination of l-lysine to produce ammonia, H2O2, and 2-keto-6-aminohexanoic acid (KH) and the majority reaction (81%) of oxidative decarboxylation of l-lysine to form CO2, H2O, and 5-aminopentanamide (5-APNM) (Fig. 1, gray panel). The oxidase activity of l-LOX/MOG could be increased by treating the enzyme with p-chloromercuribenzoate or by site-directed mutagenesis of C254I (38Matsui D. Im D.H. Sugawara A. Fukuta Y. Fushinobu S. Isobe K. Asano Y. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.FEBS Open Bio. 2014; 4 (24693490): 220-22810.1016/j.fob.2014.02.002Crossref PubMed Scopus (17) Google Scholar). However, rather than direct detection of product, the previous reports measured only the formation of ammonium in the presence and absence of amidase as a readout for the first liberation of ammonia or for the cleavage of the amide product (Fig. 1) (38Matsui D. Im D.H. Sugawara A. Fukuta Y. Fushinobu S. Isobe K. Asano Y. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.FEBS Open Bio. 2014; 4 (24693490): 220-22810.1016/j.fob.2014.02.002Crossref PubMed Scopus (17) Google Scholar, 39Isobe K. Sugawara A. Domon H. Fukuta Y. Asano Y. Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813.J. Biosci. Bioeng. 2012; 114 (22704811): 257-26110.1016/j.jbiosc.2012.04.020Crossref PubMed Scopus (21) Google Scholar). Recently, new X-ray structures of l-LOX/MOG in complex with l-lysine, l-ornithine, and l-arginine have been reported (40Im D. Matsui D. Arakawa T. Isobe K. Asano Y. Fushinobu S. Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.FEBS Open Bio. 2018; 8 (29511608): 314-32410.1002/2211-5463.12387Crossref PubMed Scopus (10) Google Scholar). Nevertheless, how l-LOX/MOG controls the two oxidase and monooxygenase activities, structurally and mechanistically, is unclear. In this study, we investigated the mechanistic features controlling the dual activities of l-LOX/MOG from Pseudomonas sp. AIU 813 by analyzing the products from the reactions of l-lysine and l-ornithine using high-resolution MS and monitoring the transient kinetics of the reactions using stopped-flow spectrophotometry. The single-turnover reactions performed under anaerobic conditions were added H2O2 under various setups to identify key catalytic features enabling decarboxylation of 5-APNM and 5-aminopentanoic acid (5-APNA). Binding interactions of l-lysine and l-ornithine with the enzyme were explored by quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations and by detection of charge transfer of the reduced FAD and the oxidized l-lysine complex by stopped-flow spectrophotometry. The common structural feature that facilitates decarboxylation of imino acids and allows the oxidases to catalyze apparent monooxygenation was also explored. To identify amino acids that can reduce the enzyme-bound oxidized FAD, a solution of l-LOX/MOG–bound oxidized FAD was added to various types of l-amino acids under anaerobic conditions. The results (Fig. S1) showed that only three l-amino acids (l-lysine, l-ornithine, and l-arginine) could reduce the enzyme-bound oxidized FAD to generate reduced FAD, which could be detected by the decrease in absorbance at 462 nm. Among the three amino acids, only the reactions of l-lysine showed complete reduction after 20 min. In contrast, l-ornithine and l-arginine showed significantly slower flavin reduction rates. Previous studies of l-LOX/MOG reported KH and 5-APNM as products from l-LOX/MOG with l-lysine and 2-keto-5-aminovaleric acid (KV) and 4-aminobutanamide (4-ABNM) as products from the reaction with l-ornithine. These data were based on HPLC identification of products and a combination of enzyme assays for both activities that measured free NH4+ before and after deamination by amide hydrolase (38Matsui D. Im D.H. Sugawara A. Fukuta Y. Fushinobu S. Isobe K. Asano Y. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.FEBS Open Bio. 2014; 4 (24693490): 220-22810.1016/j.fob.2014.02.002Crossref PubMed Scopus (17) Google Scholar). In this work, we wanted to establish and clarify whether the enzyme can generate other products in addition to these compounds and determine the ratios of compounds produced from the dual activities of l-LOX/MOG. Therefore, products from the multiple-turnover reactions of l-LOX/MOG with l-lysine and l-ornithine were identified by HPLC coupled with high-resolution MS quadrupole-time-of-flight (HPLC/QTOF-MS). Quantitative measurements were carried out by HPLC coupled with triple-quadrupole or single-quadrupole MS. Control reactions without enzyme added were also carried out to verify that the stability of the substrates was constant. Multiple-turnover reactions of l-LOX/MOG and l-lysine were carried out for 24 h, and the resulting products were analyzed. Results (Table 1) showed that all l-lysine was completely consumed, and three compounds were detected with the major product being 5-APNM (85 ± 1%), consistent with previous reports (38Matsui D. Im D.H. Sugawara A. Fukuta Y. Fushinobu S. Isobe K. Asano Y. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.FEBS Open Bio. 2014; 4 (24693490): 220-22810.1016/j.fob.2014.02.002Crossref PubMed Scopus (17) Google Scholar). However, we did not detect the expected product, KH, but detected the dehydrated form of KH (DKH) instead. Unexpectedly, we also detected a very small amount of 5-APNA (0.32 ± 0.02%). This newly detected 5-APNA may be derived from decarboxylation of KH generated in the deamination path (Figure 1, Figure 2 and Table 1). Mass spectra of all these compounds are shown in Table S1 and Fig. S2. Based on these data, it can be firmly established that l-LOX/MOG catalyzes the decarboxylation of imino lysine as a major reaction (85%) and deamination of imino lysine as a minor reaction (14.7%). HPLC chromatograms of the control and turnover reactions and all standard compounds are shown in Figs. S3–S5.Table 1Percentage of utilized substrates and product yields from multiple-turnover reactionsSubstrateCompoundsPercentagel-Lysinel-Lysine100.0 ± 0.1 (utilized)5-APNM85 ± 15-APNA0.32 ± 0.02KHDetectedDKHDetectedl-Ornithinel-Ornithine100 ± 2 (utilized)4-ABNM0.021 ± 0.0024-ABNA32.8 ± 0.2KVDetectedDKVDetected Open table in a new tab Multiple-turnover reactions of l-LOX/MOG and l-ornithine were dramatically slower than that of l-lysine (data not shown). For 24 h, all l-ornithine was consumed, and unlike the reaction of l-lysine, only 0.021 ± 0.002% of 4-ABNM from the decarboxylation of imino ornithine was detected, whereas a majority of the compounds produced was KV, as expected. Different from the previously reported data (38Matsui D. Im D.H. Sugawara A. Fukuta Y. Fushinobu S. Isobe K. Asano Y. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.FEBS Open Bio. 2014; 4 (24693490): 220-22810.1016/j.fob.2014.02.002Crossref PubMed Scopus (17) Google Scholar) but consistent with the reaction of l-lysine discussed above, a new product, 4-aminobutanoic acid (4-ABNA), resulting from decarboxylation of KV, was also detected (in significant amount, 32.8 ± 0.2%) (Fig. 3 and Table 1). Interestingly, the ratio of products from the l-ornithine reaction was different from that of l-lysine. The reaction of l-ornithine prefers to proceed via deamination to form KV and 4-ABNA more than to proceed via direct decarboxylation of imino ornithine to form 4-ABNM. Nevertheless, the decarboxylation of KV also exists. HPLC chromatograms and mass spectra of all compounds are shown in Figs. S6–S9 and Table S2. The kinetics of the l-LOX/MOG–bound oxidized FAD reduction by l-lysine and l-ornithine were investigated by monitoring absorbance changes at 462 nm (see “Experimental procedures”). The resultant kinetic traces showed that the flavin reduction by l-lysine started much quicker (0.02 s) than the reduction by l-ornithine (2 s). The enzyme-bound oxidized FAD could be completely reduced by l-lysine after 0.6 h, whereas 1.6 h was required for the reaction with l-ornithine. The kinetic traces were triphasic in both reactions of l-lysine and l-ornithine. Observed rate constants (kobs) for the reactions of l-lysine and l-ornithine are shown in Table 2. For l-lysine, kobs of the first phase is 0.540 ± 0.002 s−1, which is 22-fold greater than that of l-ornithine (0.025 ± 0.002 s−1). Therefore, the flavin reduction of l-LOX/MOG with l-lysine was dramatically faster than that of l-ornithine (Fig. 4). This indicates that l-LOX/MOG clearly prefers to use l-lysine rather than l-ornithine.Table 2kobs of the reduction of l-LOX/MOG–bound FAD by 16 mm l-lysine and l-ornithine monitored by stopped-flow spectrophotometrykobs1st phase2nd phase3rd phases−1l-Lysine0.540 ± 0.00253.2 ± 0.4 × 10−37.78 ± 0.05 × 10−3l-Ornithine0.025 ± 0.0023.18 ± 0.03 × 10−36.5 ± 0.5 × 10−4 Open table in a new tab As C4a-(hydro)peroxyflavin is a reactive intermediate commonly found in all flavin-dependent monooxygenases (32Chaiyen P. Fraaije M.W. Mattevi A. The enigmatic reaction of flavins with oxygen.Trends Biochem. Sci. 2012; 37 (22819837): 373-38010.1016/j.tibs.2012.06.005Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 41van Berkel W.J. Kamerbeek N.M. Fraaije M.W. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts.J. Biotechnol. 2006; 124 (16712999): 670-68910.1016/j.jbiotec.2006.03.044Crossref PubMed Scopus (525) Google Scholar), we investigated whether this flavin intermediate is involved in the decarboxylation reaction of l-LOX/MOG using stopped-flow spectrophotometry. Absorbance changes at 385 nm and at various other wavelengths in the regions where C4a-(hydro)peroxyflavin absorbs were monitored. Typically, when C4a-(hydro)peroxyflavin is formed, kinetic traces at 385 nm show an absorbance initial increase followed by a decrease when the intermediate eliminates H2O2 to form oxidized FAD. These kinetic traces would be clearly different from the data measured in the 450–460 nm region in which only flavin oxidation is monitored (33Pimviriyakul P. Thotsaporn K. Sucharitakul J. Chaiyen P. K" @default.
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• W3035559913 title "Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813" @default.
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