Distinct kynureninase and hydroxykynureninase activities in microorganisms: occurrence and properties of a single physiologically discrete enzyme in yeast

(i) Saccharomyces cerevisiae grown in the presence of 1.0 mM l-tryptophan slowly excreted fluorescent material that was chromatographically identifiable as 3-hydroxyanthranilate but did not excrete detectable amounts of anthranilate nor rapidly deplete the medium of l-tryptophan. Under similar growth conditions, Neurospora crassa rapidly excretes anthranilate and rapidly depletes the medium of l-tryptophan. (ii) Chromatographic analysis of crude extracts from yeast revealed a single kynureninase-type enzyme whose synthesis was not measurably affected by the presence of tryptophan in the medium. Previous studies have provided evidence for two kynureninase-type enzymes in N. crassa, an inducible kynureninase and a constitutive hydroxykynureninase. (iii) Kinetic analysis of the partially purified yeast enzyme provided Michaelis constants for l-3-hydroxykynurenine and l-kynurenine of 6.7 x 10(-6) and 5.4 x 10(-4) M, respectively. This and other kinetic properties of the yeast enzyme are comparable to those reported for the constitutive enzyme from N. crassa. (iv) These findings suggest that S. cerevisiae has in common with N. crassa the biosynthetic enzyme hydroxykynureninase but lacks the catabolic enzyme kynureninase. Therefore, it can be predicted that, unlike N. crassa, S. cerevisiae does not carry out the tryptophan-anthranilate cycle. Distinct kynureninase-type enzymes may exist in other microorganisms and in mammals.     

Inducible and constitutive kynureninases. Control of the inducible enzyme activity by transamination and inhibition of the constitutive enzyme by 3-hydroxyanthranilate.

The inducible kynureninase from Neurospora crassa is inactivated by incubation with L-alanine or L-ornithine. The inactivated enzyme is resolved to the apoenzyme by dialysis. Reactivation of the apoenzyme is achieved by incubation with pyridoxamine 5'-phosphate plus pyruvate, as well as with pyridoxal 5'-phosphate. The kynurenine hydrolysis proceeds linearly in the presence of added pyridoxal 5'-phosphate, or pyridoxamine 5'-phosphate plus pyruvate. These findings indicate that the fungal inducible kynureninase can act as an amino-transferase to control the enzyme activity, and that the control mechanism is similar to that reported for the bacterial kynureninase (Moriguchi, M. & Soda, K. (1973) Biochemistry 12, 2974-2980). The ratio of kynureninase activity to aminotransferase activity was determined with bacterial and fungal enzymes. All the inducible kynureninases from various fungal species examined are also controlled by the transamination. In contrast, the pig liver kynureninase and the fungal constitutive enzymes are little or not at all affected by preincubation with amino acids. Thus, the present regulatory mechanism does not operate in these constitutive-type enzymes. The rate of hydrolysis of L-3-hydroxykynurenine by the pig liver enzyme decreases with increase in the incubation time; the enzyme is inhibited by 3-hydroxyanthranilate produced from L-3-hydroxykynurenine. The inhibition is found in all the constitutive-type enzymes, suggesting that 3-hydroxyanthranilate plays a regulatory role in NAD biosynthesis from tryptophan.     

Isolation and characterization of a rat liver enzyme with both cysteine conjugate beta-lyase and kynureninase activity

Cysteine conjugate beta-lyase is a name applied to enzymes which cleave the S-cysteine conjugates of some xenobiotics to pyruvate, ammonia, and a thiol. Recently, several laboratories have characterized these enzymes from kidney, liver, and bacterial sources in an effort to understand their role in the genesis of novel sulfur-containing metabolites of xenobiotics and in the toxicity of some S-cysteine conjugates. Kynureninase is an enzyme which plays a key role in the biosynthesis of nicotinamide ribonucleotides. This investigation demonstrates that rat hepatic cysteine conjugate beta-lyase is the same enzyme as kynureninase. Both activities copurify on ion exchange, hydroxylapatite, and molecular exclusion chromatography. The subunit composition of enzyme prepared by two different methods is identical, Mr = 55,000. The Km values for 3-OH-kynurenine and kynurenine are 13 and 400 microM, respectively. Kynurenine and 3-hydroxykynurenine inhibit cysteine conjugate beta-lyase activity. Inactivation of the enzyme by substrates which undergo beta-elimination results in loss of kynureninase activity, but kynurenine does not inactivate the enzyme. Both enzyme activities react with anti-cysteine conjugate beta-lyase antibody. Product inhibitors of kynureninase, anthranilate, and 3-hydroxyanthranilate are also inhibitors of cysteine conjugate beta-lyase. Heat inactivation at 70 degrees C produced coincident loss of both activities. The enzyme has an absorption maximum at 432 nm suggesting a bound pyridoxal phosphate. These data show that at least one cysteine conjugate beta-lyase is a pyridoxal phosphate enzyme with a biological function other than xenobiotic metabolism. The enzyme can catalyze two distinct types of reactions, i.e. beta-elimination and the kynureninase reaction.     

Kynureninase-type enzymes and the evolution of the aerobic tryptophan-to-nicotinamide adenine dinucleotide pathway.

Kynureninase-type (L-kynurenine hydrolase, EC 3.7.1.3) activity has been found to be present in the livers of fish, amphibia, reptiles, and birds. In addition to past information concerning this enzyme activity in mammalian liver, it is now clear that all the major classes of vertebrates carry a highly specialized kynureninase-type enzyme, which we have termed a hydroxykynureninase. To compare the reactivities of these enzymes with L-kynurenine and L-3-hydroxykynurenine, ratios of tau values (Km/V) were used. Based on this comparison, the bacterium Pseudomonas fluorescens carries the most efficient kynureninase, whereas the amphibian Xenopus laevis has the most efficient hydroxykynureniase. In these two cases, the ratio of tau values differs by a factor of 38 000. It is hypothesized that the tryptophan-to-nicotinamide adenine dinucleotide biosynthetic pathway evolved from a catabolic system of enzymes, and that the differences observed in the kynureninase-type enzymes between lower and higher organisms reflect the specialization of the function of these enzymes from a strictly catabolic role to an anabolic one during the course of evolution.     

Comparison of inducible and constitutive kynureninases of Neurospora crassa.

Two types of kynureninase were isolated from Neurospora crassa IFO 6068. The formation of one of them, which was separated from the inducible kynureninase by DEAE-cellulose chromatography, was independent of the presence of tryptophan in the growth medium. Ouchterlony double-diffusion analysis and immunochemical titration indicated that the constitutive-type enzyme is immunologically different from the inducible enzyme. We confirmed by a selective assay method with antiserum that the addition of tryptophan to the medium does not affect the formation of one of the enzymes (constitutive-type). The constitutive kynureninase was purified approximately 650-fold and was free of the inducible enzyme as judged by analytical gel electrophoresis. The molecular weight and optimum pH values of both enzymes are very similar. However, the constitutive enzyme shows much higher activity and affinity for L-3-hydroxykynurenine than for L-kynurenine, suggesting that the enzyme functions biosynthetically as a 3-hydroxykynureninase. Constitutive kynureninase activities were widely found in all the fungi tested, whereas the inducible enzyme activity was not present in Mucor or Rhizopus species. The inducible enzymes of all the Neurospora strains examined were shown to be immunologically identical.     

Regulation of the Pathway for the Degradation of Anthranilate in Aspergillus niger

Studies were carried out to determine the factors governing the induction of anthranilate hydroxylase and other enzymes in the pathway for the dissimilation of anthranilate by Aspergillus niger (UBC 814). The enzyme was induced by growth in the presence of tryptophan, kynurenine, anthranilate, and, surprisingly, by 3-hydroxyanthranilate, which was not an intermediate in the conversion of anthranilate to 2,3-dihydroxybenzoate. There was an initial lag in the synthesis of anthranilate hydroxylase when induced by tryptophan, anthranilate, and 3-hydroxyanthranilate. Cycloheximide inhibited the enzyme induction. Comparative studies on anthranilate hydroxylase, 2,3-dihydroxybenzoate carboxy-lyase, and catechol 1:2-oxygenase revealed that these enzymes were not coordinately induced by either anthranilate or 3-hydroxyanthranilate. Structural requirements for the induction of anthranilate hydroxylase were determined by using various analogues of anthranilate. The activity of the constitutive catechol oxygenase was increased threefold by exposure to anthranilate, 2,3-dihydroxybenzoate, or catechol. 3-Hydroxyanthranilate did not enhance the levels of catechol oxygenase activity.     

Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate in the final enzymatic step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide. The enzyme requires Fe2+ as a cofactor and is inactivated by 4-chloro-3-hydroxyanthranilate. HAD from Ralstonia metallidurans was crystallized, and the structure was determined at 1.9 A resolution. The structures of HAD complexed with the inhibitor 4-chloro-3-hydroxyanthranilic acid and either molecular oxygen or nitric oxide were determined at 2.0 A resolution, and the structure of HAD complexed with 3-hydroxyanthranilate was determined at 3.2 A resolution. HAD is a homodimer with a subunit topology that is characteristic of the cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A. On the basis of the crystal structures of HAD, mutagenesis studies were carried out in order to elucidate the enzyme mechanism. In addition, a new mechanism for the enzyme inactivation by 4-chloro-3-hydroxyanthranilate is proposed.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

Kynureninase-Type enzymes of Penicillum roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens: further evidence for distinct kynureninase and hydroxykynureninase activities.

The kynureninase-type enzymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalyze the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhizopus stolonifer was found to contain a single, constitutive enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 times 10-minus 6 and 2.5 times 10-minus 4 M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enzyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 times 10-minus 5 to 14.3 times 10-minus 5 M, as well as a constitutive enzyme with Km for the two substrates of similar to 4 times 10-minus 6 M and 10-minus 4 M. The bacterium Pseudomonas fluorescens has a single, inducible enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 times 10-minus 4 and 7 times 10-minus 5 M. In addition, significant differences in maximal velocities (Vmax) were observed in two cases. The Vmax of the inducible activity from P. fluorescens was 4.5 times greater for L-kynurenine than L-3-hydroxykynurenine, whereas the Vmax of the constitutive activity from R. stolonifer was 2.5 times greater for L-3-hydroxykynurenine. It is concluded (i) that the constitutive activities are hydroxykynureninases involved in the biosynthesis of nicotinamide adenine dinucleotide from L-tryptophan, (ii) that the inducible activities are kynureninases involved in the catabolism of L-tryptophan to anthranilate, and (iii) that R. stolonifer and P. fluorescens, respectively, carry the most specific examples of each type of enzyme.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders. Here, we describe the mechanism by which 4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that 4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of oxygen and the production of superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated enzyme demonstrated that the inhibitor did not form an adduct with the enzyme and that four conserved cysteines were oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site.     

Purification and Characterization of the Two 6-Phosphogluconate Dehydrogenase Species from Pseudomonas multivorans

The two species of 6-phosphogluconate dehydrogenase (EC 1.1.1.43) from Pseudomonas multivorans were resolved from extracts of gluconate-grown bacteria and purified to homogeneity. Each enzyme comprised between 0.1 and 0.2% of the total cellular protein. Separation of the two enzymes, one which is specific for nicotinamide adenine dinucleotide phosphate and the other which is active with nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate was facilitated by the marked difference in their respective isoelectric points, which were at pH 5.0 and 6.9. Comparison of the subunit compositions of the two enzymes indicated that they do not share common peptide chains. The enzyme active with nicotinamide adenine dinucleotide was composed of two subunits of about 40,000 molecular weight, and the nicotinamide adenine dinucleotide phosphate-specific enzyme was composed of two subunits of about 60,000 molecular weight. Immunological studies indicated that the two enzymes do not share common antigenic determinants. Reduced nicotinamide adenine dinucleotide phosphate strongly inhibited the 6-phosphogluconate dehydrogenase active with nicotinamide adenine dinucleotide by decreasing its affinity for 6-phosphogluconate. Guanosine-5'-triphosphate had a similar influence on the nicotinamide adenine dinucleotide phosphate-specific 6-phosphogluconate dehydrogenase. These results in conjunction with other data indicating that reduced nicotinamide adenine dinucleotide phosphate stimulates the conversion of 6-phosphogluconate to pyruvate by crude bacterial extracts suggest that in P. multivorans, the relative distribution of 6-phosphogluconate into the pentose phosphate and Entner-Doudoroff pathways might be determined by the intracellular concentrations of reduced nicotinamide adenine dinucleotide phosphate and purine nucleotides.     

Kynurenine metabolism in vitamin-B-6-deficient rat liver after tryptophan injection.

Tryptophan contents of liver, serum and kidney were determined in normal and vitamin-B-6-deficient rats after tryptophan injection. Tryptophan contents of normal and B-6-deficient liver were different, but not those in serum and kidney. Both kynurenine and 3-hydroxykynurenine accumulated in B-6-deficient liver more than in the normal. The 3-hydroxykynurenine contents after tryptophan injection (30 mg/100 g body wt.) increased to 1380 nmol/g of liver at 1-1.5 h, a value sufficient to produce xanthurenate, in view of the Km value of kynurenine aminotransferase. The enzymes metabolizing kynurenine were assayed at various times after tryptophan injection. The activity of kynureninase holoenzyme in B-6-deficient liver was much decreased, but the activity of total enzyme was not changed. It appeared that a high dose of tryptophan in B-6-deficient rats could cause a greater deficiency of pyridoxal 5-phosphate. Tryptophan metabolism in B-6-deficient rat liver after tryptophan administration is discussed.     

Urine 3-hydroxykynurenine is higher during the postovulatory phase than in the preovulatory phase indicating a higher vitamin B6 requirement

The relationship between l-tryptophan to nicotinamide metabolism and the menstrual cycle of Japanese women was investigated. Nine metabolism intermediates from urine samples collected during the preovulatory and postovulatory phases were measured. Only urine 3-hydroxykynurenine was higher in the postovulatory phase than in the preovulatory phase. This increase in 3-hydroxykynurenine suggests a decreased reaction of 3-hydroxykynurenine?→?3-hydroxyanthranilic acid catalyzed by kynureninase, a vitamin B₆ enzyme.     

Determination of the hydride transfer stereospecificity of nicotinamide adenine dinucleotide linked oxidoreductases by proton magnetic resonance.

A facile proton magnetic resonance technique is described for the determination of the coenzyme stereospecificity during hydride transfer reactions catalyzed by pyridine nucleotide dependent oxidoreductases. The reliability of this technique was demonstrated by examining the coenzyme stereospecificity of lactate, malate, and 3-phosphoglycerate dehydrogenases, which are known to be A-stereospecific enzymes, as well as triosephosphate and octopine dehydrogenases, which are known to be B-stereospecific enzymes. Furthermore, by applying this technique, it was shown that the previously unstudied enzymes D-beta-hydroxybutyrate and 4-aminobutanal dehydrogenases are B- and A-stereospecific enzymes, respectively. In addition, the nicotinamide adenine dinucleotide linked reaction of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was found to be B stereospecific, like the reaction of the nicotinamide adenine dinucleotide phosphate linked yeast enzyme.     

Amino-acid sensing and degrading pathways in immune regulation

Indoleamine 2,3-dioxygenases (IDOs) − belonging in the heme dioxygenase family and degrading tryptophan − are responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD⁺). As such, they are expressed by a variety of invertebrate and vertebrate species. In mammals, IDO1 has remarkably evolved to expand its functions, so to become a prominent homeostatic regulator, capable of modulating infection and immunity in multiple ways, including local tryptophan deprivation, production of biologically active tryptophan catabolites, and non-enzymatic cell-signaling activity. Much like IDO1, arginase 1 (Arg1) is an immunoregulatory enzyme that catalyzes the degradation of arginine. Here, we discuss the possible role of amino-acid degradation as related to the evolution of the immune systems and how the functions of those enzymes are linked by an entwined pathway selected by phylogenesis to meet the newly arising needs imposed by an evolving environment.     

A nuclear magnetic resonance-based functional assay for nicotinamide adenine dinucleotide synthetase

Nicotinamide adenine dinucleotide synthetase (NadE) is an essential enzyme for bacterial pathogens and is thus a promising antibacterial target. It catalyzes the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. Changes in chemical shifts that occur in the nicotinic acid ring as it is converted to nicotinamide can be used for monitoring the reaction. A robust nuclear magnetic resonance-based activity assay was developed using robotically controlled reaction initiation and quenching. The single-enzyme assay has less potential for false positives compared to a coupled activity assay and is especially well suited to the high concentration of compounds in fragment screens. The assay has been used to screen fragment libraries for NadE inhibitors.     

Involvement of 4-hydroxymandelic acid in the degradation of mandelic acid by Pseudomonas convexa.

A microorganism capable of degrading DL-mandelic acid was isolated from sewage sediment of enrichment culture and was identified as Pseudomonas convexa. It was found to metabolize mandelic acid by a new pathway involving 4-hydroxymandelic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, and 3,4-dihydroxybenzoic acid as aromatic intermediates. All the enzymes of the pathway were demonstrated in cell-free extracts. L-Mandelate-4-hydroxylase, a soluble enzyme, requires tetrahydropteridine, nicotinamide adenine dinucleotide phosphate, reduced form, and Fe2+ for its activity. The next enzyme, L-4-hydroxymandelate oxidase (decarboxylating), a particulate enzyme, requires flavine adenine dinucleotide and Mn2+ for its activity. A nicotinamide adenine dinucleotide-dependent, as well as a nicotinamide adenine dinucleotide phosphate-dependent, benzaldehyde dehydrogenase has been resolved and partially purified.     

Crystal structure of Homo sapiens kynureninase

Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.