Biomedical subjects

J R Andreesen

Publications and source records attributed to J R Andreesen.

At least 37 records · Page 2Linked to original sources

PubMed1999

Selenium-containing xanthine dehydrogenase from Eubacterium barkeri.

A specific dehydrogenase, different from nicotinic acid hydroxylase, was induced during growth of Eubacterium barkeri on xanthine. The protein designated as xanthine dehydrogenase was enriched 39-fold to apparent homogeneity using a three-step purification scheme. It exhibited an NADP-dependent specific activity of 164 micromol xanthine oxidized per min and per mg of protein. In addition it showed an NADPH-dependent oxidase and diaphorase activity. A molecular mass of 530 kDa was determined for the native enzyme and SDS/PAGE revealed three types of subunits with molecular masses of 17.5, 30 and 81 kDa indicating a dodecameric native structure. Molybdopterin was identified as the molybdenum-complexing cofactor using activity reconstitution experiments and fluorescence measurements after KI/I2 oxidation. The molecular mass of the cofactor indicated that it is of the dinucleotide type. The enzyme contained iron, acid-labile sulfur, molybdenum, tungsten, selenium and FAD at molar ratios of 17.5, 18.4, 2.3, 1.1, 0.95 and 2.8 per mol of native enzyme. Xanthine dehydrogenase was inactivated upon incubation with arsenite, cyanide and different purine analogs. Reconstitution experiments of xanthine dehydrogenase activity by addition of selenide and selenite performed with cyanide-inactivated enzyme and with chloramphenicol-treated cells, respectively, indicated that selenium is not attached to the protein in a covalently bound form such as selenocysteine.

Animals↗

PubMed1998

2-Hydroxyisonicotinate dehydrogenase isolated from Mycobacterium sp. INA1.

2-Hydroxyisonicotinate dehydrogenase from Mycobacterium sp. INA1 was purified 26-fold to apparent homogeneity. The enzyme is involved in isonicotinate degradation by Mycobacterium sp. INA1 and catalyzes the conversion of 2-hydroxyisonicotinate to 2,6-dihydroxypyridine-4-carboxylate. The purified protein exhibited a native molecular mass of 300 kDa and subunits of 97, 31 and 17 kDa, respectively, indicating an alpha 2 beta 2 gamma 2 structure. The absorption spectrum of the homogeneous enzyme was characteristic for an iron/sulfur flavoprotein, 3.8 mol of iron, 3.7 mol of acid labile sulfur, 0.94 mol of FAD and 0.75 mol of molybdenum were determined per mol of protomer. The molybdenum cofactor was identified as molybdopterin cytosine dinucleotide. 2-Hydroxyisonicotinate dehydrogenase was inactivated in the presence of cyanide. According to these basic properties the protein seems to belong to the class of molybdo-iron/sulfur flavoproteins of the xanthine oxidase family.

Amino Acid Sequence↗

PubMed1998

Partial purification of an iron-dependent L-serine dehydratase from Clostridium sticklandii.

An oxygen-sensitive and highly unstable L-serine dehydratase was partially purified from the Gram-positive anaerobe Clostridium sticklandii. The final active preparation contained five proteins of 27, 30, 44.5, 46, and 58 kDa as judged by SDS-PAGE. The N-terminal sequence of the 30 kDa subunit showed some similarity to the alpha-subunits of the iron-containing L-serine dehydratases from Clostridium propionicum and Peptostreptococcus asaccharolyticus. Oxygen-inactivated L-serine dehydratase from C. sticklandii was reactivated by incubation with Fe2+ under reducing conditions. Furthermore, the enzyme was inactivated by iron-chelating substances like phenanthroline and EDTA. Pyridoxal-5-phosphate (PLP) did not stimulate the activity, and known inhibitors of PLP-containing enzymes such as NaBH4 had no effect on the activity of L-serine dehydratase from C. sticklandii.

Aerobiosis↗

PubMed1998

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components.

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.

Amino Acid Oxidoreductases↗

PubMed1998

Tissierella creatinophila sp. nov., a gram-positive, anaerobic, non-spore-forming, creatinine-fermenting organism.

A strictly anaerobic, Gram-positive, non-spore-forming bacterium was isolated from sewage sludge which grew on creatinine as sole source of carbon and energy. This new isolate, designated strain KRE 4T, totally degraded creatinine via creatine, sarcosine and glycine to the products acetate, monomethylamine, ammonia and carbon dioxide. Growth on creatinine or creatine was selenium-dependent and stimulated by formate, indicating the involvement of a creatine reductase, sarcosine reductase and/or glycine reductase. This was substantiated by the fact that creatine, sarcosine and glycine were reduced by cell-free extracts. Growth on creatinine or creatine was also possible in the absence of formate, but with an increase in doubling time. The new bacterium occurred as rod-shaped cells, which exhibited an angular form (2-6 microns long and 0.7-1.1 microns wide) and showed motility by means of peritrichous flagella. The G+C content of the DNA was 30 mol %. Comparative 16S rRNA sequence analysis demonstrated that strain KRE 4T represents a new subline within the genus Tissierella. Due to its very restricted substrate spectrum and the inability of whole cells to utilize sarcosine and glycine as intermediates of creatine breakdown, this organism can be readily separated from currently described species of Tissierella. Therefore, based on the phenotypic and phylogenetic distinctiveness of the new isolate, it si proposed that the bacterium be classified as a new species of the genus Tissierella, Tissierella creatinophila sp. nov. The type strain is KRE 4 (= DSM 6911T).

Base Sequence↗

PubMed1997

Two-component flavin-dependent pyrrole-2-carboxylate monooxygenase from Rhodococcus sp.

Pyrrole-2-carboxylate can serve as the sole source of carbon, nitrogen, and energy for a strain tentatively identified to belong to the genus Rhodococcus. An NADH-dependent oxygenase activity was detected in cell extracts that initiated the degradation of the substrate. During purification of the enzyme, this activity was separated into two protein components which were both purified to apparent homogeneity. A small monomeric 18.7-kDa protein designated as reductase, catalyzed in vitro the NADH and FAD-dependent reduction of cytochrome c and had an NADH-oxidase activity. The second component, a 54-kDa protein with a trimeric native structure had no enzymatic activity by itself, but exhibited a pyrrole-2-carboxylate-dependent oxygen consumption when it was complemented with the reductase component, FAD, and NADH. This indicated that the large protein referred to as oxygenase was responsible for the oxygen-dependent hydroxylation of the substrate. The rate of an uncoupled NADH oxidation without hydroxylation of the substrate was found to be strongly dependent on the molar ratio of both components. The uncoupling was nearly completely suppressed by a 5-7-fold molar excess of the oxygenase component. The small protein was N-terminally blocked. It was thus proteolytically digested and four of the resulting peptides were sequenced comprising 47 amino acids. The sequences of these fragments were similar to the sequences reported for the small component of different two-component flavin monooxygenases. Furthermore, the N-terminus of the oxygenase component showed high sequence similarity to the second, usually large subunit of these enzymes and to two single-component flavin monooxygenases. Thus, the enzyme from Rhodococcus sp. designated as pyrrole-2-carboxylate monooxygenase belongs to the recently discovered new class of two-component flavin aromatic monooxygenases. Some of the basic properties of both components were determined and their interaction during catalysis was investigated.

Amino Acid Sequence↗

PubMed1997

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale.

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale.

Amino Acid Sequence↗

PubMed1997

Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase.

An organism tentatively identified as Ralstonia eutropha was isolated from enrichment cultures containing tetrahydrofurfuryl alcohol (THFA) as the sole source of carbon and energy. The strain was able to tolerate up to 200 mM THFA in mineral salt medium. The degradation was initiated by an inducible ferricyanide-dependent alcohol dehydrogenase (ADH) which was detected in the soluble fraction of cell extracts. The enzyme catalyzed the oxidation of THFA to the corresponding tetrahydrofuran-2-carboxylic acid. Studies with n-pentanol as the substrate revealed that the corresponding aldehyde was released as a free intermediate. The enzyme was purified 211-fold to apparent homogeneity and could be identified as a quinohemoprotein containing one pyrroloquinoline quinone and one covalently bound heme c per monomer. It was a monomer of 73 kDa and had an isoelectric point of 9.1. A broad substrate spectrum was obtained for the enzyme, which converted different primary alcohols, starting from C2 compounds, secondary alcohols, diols, polyethylene glycol 6000, and aldehydes, including formaldehyde. A sequence identity of 65% with a quinohemoprotein ADH from Comamonas testosteroni was found by comparing 36 N-terminal amino acids. The ferricyanide-dependent ADH activity was induced during growth on different alcohols except ethanol. In addition to this activity, an NAD-dependent ADH was present depending on the alcohol used as the carbon source.

Alcaligenes↗

PubMed1996

Studies on the inactivation of the flavoprotein D-amino acid oxidase from Trigonopsis variabilis.

Inactivation of D-amino acid oxidase occurred by different mechanisms. The enzyme showed a rapid loss of activity in the presence of micromolar amounts of Cu2+ and Hg2+. It was also sensitive to oxidative inactivation by Fe2+ and H2O2 when both reagents were added in millimolar amounts. When oxidatively inactivated D-amino acid oxidase and a corresponding non-treated control were modified with the sulfhydryl-modifying, fluorescent reagent monobromobimane and subsequently digested with endoproteinase Glu-C, Cys-298 was identified to be a target for oxidative modification according to differences in the known peptide profile of fluorescence intensity. Another reason for the observed loss of enzyme activity in crude extracts was the specific proteolytic digestion of D-amino acid oxidase, which was dependent on the growth phase of the cells used. This cleavage was catalyzed by a serine-type proteinase and was the introductory step for the further complete degradation of the enzyme. In addition, a coenriched 50-kDa protein, identified as NADPH-specific glutamate dehydrogenase, significantly decreased the stability of the D-amino acid oxidase activity. Treatment of apo-D-amino acid oxidase from T. variabilis with monobromobimane resulted in a significantly increased fluorescence of two peptides, neither of which contained any cysteine residue. Thus, an involvement of cysteine residues in binding the FAD coenzyme should be excluded.

Amino Acid Sequence↗

PubMed1995

Purification and characterization of protein PB of betaine reductase and its relationship to the corresponding proteins glycine reductase and sarcosine reductase from Eubacterium acidaminophilum.

Simple complementation assay systems were developed for the substrate-specific proteins PB of glycine reductase, sarcosine reductase, and betaine reductase, in which acetyl phosphate was detected as the product in all three cases. The betaine-specific subunits of protein B (PB betaine) responsible for betaine reductase activity were purified to homogeneity from cells of Eubacterium acidaminophilum. The molecular masses of the two different subunits were 45 kDa and 48 kDa according to SDS/PAGE. The molecular mass of the native protein was about 200 kDa, indicating and alpha 2 beta 2 structure. The glycine-specific protein B (PB glycine) was partially purified and subunits of 47 kDa and 27 kDa were N-terminally sequenced. The latter subunits cross-reacted with antibodies raised against PB betaine and showed high sequence similarity to the 45-kDa and 48-kDa subunits of PB betaine, respectively. [2-14C]Glycine could be covalently coupled to the 47-kDa subunit by treatment with borohydride. By the same procedure, [2-14C]sarcosine labeled a protein of the same size. Like the sarcosine reductase activity, this protein was not present in glycine-grown cells, indicating its specific involvement in sarcosine metabolism. The labile viologen-dependent formate dehydrogenase purified with the respective PB proteins and could be tentatively assigned to a 95-kDa protein.

Amino Acid Oxidoreductases↗

PubMed1995

Glycine reductase of Clostridium litorale. Cloning, sequencing, and molecular analysis of the grdAB operon that contains two in-frame TGA codons for selenium incorporation.

A 2.8-kb HindIII fragment, containing three open reading frames, has been cloned and sequenced from Clostridium litorale. The first gene grdA encoded the selenocysteine-containing protein PA of the glycine reductase complex, a protein of 159 amino acids with a deduced molecular mass of 16.7 kDa. The second gene (grdB) encoded the 47-kDa subunit of the substrate-specific selenoprotein PB glycine that is composed of 437 amino acids. The third gene contained the 5'-region of the gene for thioredoxin reductase, trxB. All gene products shared high similarity with the corresponding proteins from Eubacterium acidaminophilum. In both genes grdA and grdB, the opal termination codon (TGA) was found inframe, indicating the presence of selenocysteine in both polypeptides. Northern-blot analysis showed that grdA and grdB are organized as one operon. Unlike Escherichia coli, no stable secondary structures of the corresponding mRNA were found immediately downstream of the UGA codons to direct an insertion of selenocysteine into the grdA and grdB transcripts of C. litorale. Instead, a secondary structure was identified in the 3'-untranslated region of grdB.

Amino Acid Oxidoreductases↗

PubMed1995

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii.

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units.mg-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only L-threo-threonine, DL-beta-hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent Km values for L-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn2+, Co2+ and Cu2+ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.

Alcohol Oxidoreductases↗

PubMed1994

Glycine metabolism in anaerobes.

Some strict anaerobic bacteria catalyze with glycine as substrate an internal Stickland reaction by which glycine serves as electron donor being oxidized by glycine-cleavage system or as electron acceptor being reduced by glycine reductase. In both cases, energy is conserved by substrate level phosphorylation. Except for the different substrate-activating proteins PB, reduction of sarcosine or betaine to acetyl phosphate involves in Eubacterium acidaminophilum the same set of proteins as observed for glycine, e.g. a unique thioredoxin system as electron donor and an acetyl phosphate-forming protein PC interacting with the intermediarily formed Secarboxymethylselenoether bound to protein PA.

Amino Acid Oxidoreductases↗

PubMed1994

Purification and characterization of a pyrrole-2-carboxylate oxygenase from Arthrobacter strain Py1.

Pyrrole-2-carboxylate oxygenase was purified 8.2-fold to homogeneity from Arthrobacter strain Py1 grown on pyrrole-2-carboxylate as sole carbon, nitrogen, and energy source. FAD and dithioerythritol had to be present during the purification procedure to stabilize the enzyme activity. The molecular mass of the pyrrole-2-carboxylate oxygenase was about 160 kDa by gel filtration chromatography and native gradient PAGE, only one polypeptide of about 60 kDa was present after SDS-PAGE. The FAD content was 2.7 to 3.6 mol FAD per enzyme (160 kDa). The non-covalently bound FAD of the pyrrole-2-carboxylate oxygenase was reduced by NADH and reoxidized by oxygen and pyrrole-2-carboxylate. The enzyme exhibited a narrow substrate specificity. Besides pyrrole-2-carboxylate, only pyrrole, pyrrole-2-aldehyde, and indole-2-carboxylate stimulated the oxygen consumption at a very low rate. The enzyme activity was strongly reduced by different sulfhydryl group inhibitors, but it could be restored by 2-mercaptoethanol or dithiothreitol. The content of pyrrole-2-carboxylate oxygenase was about 6% of the soluble protein as determined by antibodies raised against the enzyme. No cross reacting material was present in other bacteria also able to degrade pyrrole-2-carboxylate. A low amount of the enzyme was present in uninduced cells of Arthrobacter strain Py1, although the enzymatic activity was below the detection limit. The N-terminal amino acid sequence of the enzyme did not contain the consensus sequence GXGXXG found to be present close to the N-terminus of many flavin-dependent monoxygenases sequenced so far.

Amino Acid Sequence↗

PubMed1993

Evidence for the functional importance of Cys298 in D-amino acid oxidase from Trigonopsis variabilis.

D-Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freeze/thawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio A280/A450 of about 6.5 and was obtained in high yield (63%) and a good stability. Using D-methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26 degrees C, corresponding to 185 U/mg polarographically at 37 degrees C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39-kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27-kDa and 12-kDa fragments as revealed by SDS/PAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl-modifying reagents tested. Inactivation by 5,5'-dithiobis(-2-nitrobenzoate) was correlated with a modification of up to 2 mol/mol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl-modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reverse-phase chromatography of endoproteinase Glu-C-digested monobromobimane-labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue.

Amino Acid Sequence↗

PubMed1993

Components of glycine reductase from Eubacterium acidaminophilum. Cloning, sequencing and identification of the genes for thioredoxin reductase, thioredoxin and selenoprotein PA.

The genes encoding thioredoxin reductase (trxB), thioredoxin (trxA), protein PA of glycine reductase (grdA) and the first 23 amino acids of the large subunit of protein PC of glycine reductase (grdC) belonging to the reductive deamination systems present in Eubacterium acidaminophilum were cloned and sequenced. The proteins were products of closely linked genes with 314 codons (thioredoxin reductase), 110 codons (thioredoxin), and 158 codons (protein PA). The protein previously called 'atypically small lipoamide dehydrogenase' or 'electron transferring flavoprotein' could now conclusively be identified as a thioredoxin reductase (subunit mass of 34781 Da) by the alignment with the enzyme of Escherichia coli showing the same typical order of the corresponding domains. The thioredoxin (molecular mass of 11742 Da) deviated considerably from the known consensus sequence, even in the most strongly conserved redox-active segment WCGPC that was now GCVPC. The selenocysteine of protein PA (molecular mass of 16609 Da) was encoded by TGA. The protein was highly similar to those of Clostridium purinolyticum and Clostridium sticklandii involved in glycine reductase. Thioredoxin reductase and thioredoxin of E. acidaminophilum could be successfully expressed in E. coli.

Amino Acid Oxidoreductases↗

PubMed1993

Catabolism of isonicotinate by Mycobacterium sp. INA1: extended description of the pathway and purification of the molybdoenzyme isonicotinate dehydrogenase.

Catabolism of isonicotinate by Mycobacterium sp. INA1 has been shown to proceed via 2-hydroxyisonicotinate, 2,6-dihydroxyisonicotinate (citrazinate), citrazyl-CoA and 2,6-dioxopiperidine-4-carboxyl-CoA. An extended pathway involving propane-1,2,3-tricarboxylate as a further intermediate is presented in this paper. Propane-1,2,3-tricarboxylate was oxidized stepwise to 2-oxoglutarate involving an oxidase, aconitase and isocitrate dehydrogenase. Isonicotinate dehydrogenase catalyses the first step of isonicotinate metabolism in Mycobacterium sp. INA1. The enzyme was purified to apparent homogeneity by a three-step procedure. Enrichment was accompanied by partial loss in specific activity. The native enzyme had a molecular mass of either 125 kDa or 250 kDa, when estimated by native gradient PAGE or gel filtration, respectively. SDS-gel electrophoresis revealed three types of subunits with molecular masses of approximately 83, 31 and 19 kDa. N-Terminal amino acid sequences of all three subunits have been determined. Molybdenum, iron, acid-labile sulphur and FAD were present at molar ratios of 1, 4, 4, 1 per protomer (125 kDa). The molybdenum-complexing cofactor was shown to be molybdopterin cytosine dinucleotide. Besides isonicotinate, only quinoline-4-carboxylate was found to be oxidized at appreciable rates.

Amino Acid Sequence↗

PubMed1992

Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum.

Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl-Sepharose and Sepharose S. The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an alpha 4 beta 4 composition. The smaller subunit was identified as an acetyl-group-transferring protein, the 57-kDa protein was hydrophobic. N-terminal amino acid sequences were determined for both subunits. Antibodies raised against the 48-kDa subunit showed cross-reactions with extracts of E. acidaminophilum grown on different substrates and with extracts from other glycine-utilizing anaerobic bacteria such as Clostridium purinolyticum, C. sticklandii, and C. sporogenes. The respective protein from the former two organisms corresponded in molecular mass. When protein PA was chemically carboxymethylated by iodo[2-14C]acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E. acidaminophilum.

Amino Acid Oxidoreductases↗