Purification and biochemical characterization of some of the properties of recombinant human kynureninase Harold A. Walsh and Nigel P. Botting School of Chemistry, University of St Andrews, St Andrews, Fife, Scotland, UK Recombinant human kynureninase ( L -kynurenine hydrol- ase, EC 3.7.1.3) was purified to homogeneity (60-fold) from Spodoptera frugiperda (Sf9) cells infected with baculovirus containing the kynureninase gene. The purification protocol comprised ammonium sulfate precipitation and several chromatographic steps, including DEAE–Sepharose CL-6B, hydroxyapatite, strong anionic and cationic sepa- rations. The purity of the enzyme was determined by SDS/ PAGE, and the molecular mass verified by MALDI-TOF MS. The monomeric molecular mass of 52.4 kDa deter- mined was > 99.99% of the predicted molecular mass. A UV absorption spectrum of the holoenzyme resulted in a peak at 432 nm. The optimum pH was 8.25 and the enzyme displayed a strong dependence on the ionic strength of the buffer for optimum activity. This cloned enzyme was highly specific for 3-hydroxykynurenine (K m ¼ 3.0 l M ±0.10) and was inhibited by L -kynurenine (K i ¼ 20 l M ), D -kynurenine (K i ¼ 12 l M ) and a synthetic substrate analogue D , L -3,7-dihydroxydesaminokynurenine (K i ¼ 100 n M ). The activity/concentration profile for kynureninase from this source was sigmoidal in all instances. There appeared to be partial inhibition by substrate, and excess pyridoxal 5¢-phosphate was found to be inhibitory. Keywords: kynureninase; kynurenine; neuroprotection; quinolinic acid; tryptophan metabolism. Rapid progress in the pathophysiology of human diseases has always been hampered by the availability of human tissue, aesthetics, and ethical considerations. The principle aim of this study was to express a clone of human kynureninase in an appropriate host that would yield sufficient quantities of protein to permit identification and biochemical characterization of the enzyme and investiga- tion into the effects of various synthetic and endogenous inhibitors. Achieving these objectives could provide an avenue for pharmacological modulation of the synthesis of the N-methyl D -aspartate receptor agonist and the excito- toxin, quinolinic acid, in addition to elevating the levels of the neuroprotective kynurenate [1]. Quinolinic acid has been implicated as an aetiological factor in a range of neurode- generative diseases which include epilepsy, Huntington’s disease, AIDS-related dementia, and septicaemia, where it is released as part of the inflammatory response to injury [2,3]. Kynureninase is one of the enzymes involved in the tryptophan metabolic pathway. It is a pyridoxal 5¢-phos- phate (PLP)-dependent enzyme which catalyses the b,c- hydrolytic cleavage of the amino acids L -kynurenine (1)and L -3-hydroxykynurenine (2)togive L -alanine (3) and either anthranilic acid (4) or 3-hydroxyanthranilic acid (5) (Scheme 1) [4]. This pathway is crucial in the biosynthesis of nicotinamide nucleotides [5] and also gives rise to other pathophysiologically important compounds such as picolinic acid, an enhancer of nitric oxide synthase expression [6]. Kynureninase has been purified and characterized from a number of different sources, such as bacteria, vertebrates and fungi, but very little is known about the human form. The microbial enzyme from some sources has been shown to be present as two isozymes with differing specificities toward L -kynurenine and 3-hydroxykynurenine [7]. Recom- binant human enzyme is reported to be a homodimer with a monomeric molecular mass of  52.4 kDa and shares an amino-acid sequence homology of about 85% with cyto- solic rat hepatic kynureninase, which has also been cloned and expressed [8]. There have been a few attempts to isolate and purify cloned human kynureninase but with limited success, although the bacterial enzyme has been cloned [9]. Previous researchers [10] have demonstrated activity in human embryonic kidney fibroblast (HEK-293)-transfected cell homogenates with a K m of 13.2 l M for 3-hydroxyky- nurenine and 671 l M for L -kynurenine for the catalytically active human enzyme. MATERIALS AND METHODS Materials All chemicals (reagent grade) were purchased from Sigma except for the ion exchangers and the Affi-Blue gel, which were obtained from Bio-Rad. The nitrocellulose filters used for concentration and buffer equilibration of active enzyme fractions were purchased from Millipore (UK). Protein expression A cDNA clone encoding human liver kynureninase was a gift from Dr Andrea Cesura, Hoffman la Roche. The cDNA was isolated by the method of 1 Alberati-Giani et al. Correspondence to N. P. Botting, School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland, UK. Fax: + 44 1334 463808, Tel.: + 44 1334 463856, E-mail: npb@st-andrews.ac.uk Abbreviations: PLP, pyridoxal 5¢-phosphate. Enzyme: kynureninase ( L -kynurenine hydrolase, EC 3.7.1.3). 7 (Received 27 November 2001, revised 25 February 2002, accepted 25 February 2002) Eur. J. Biochem. 269, 2069–2074 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02854.x [10] and cloned into the ÔBac-to-BacÕ Baculovirus Expres- sion System (Gibco-BRL) which was used to express kynureninase in Spodoptera frugiperda (Sf9) insect cells [11]. Sf9 cells were grown at 28 °C in TC-100 suspension cultures of 10 L containing a minimal amount of fetal calf serum (1–2%) until 10 mL of growing cells resulted in a confluent layer when placed in a small Petri dish. Infection of the Sf9 cells and expression of kynureninase proceeded in TC-100 in the absence of any fetal calf serum. The infection was allowed to proceed for 96 ± 12 h depending on the degree of lysis. Light microscopy was used to monitor the infection process, and, when isolated nuclei appeared amid a host of grossly deformed cells, the cells were harvested and active enzyme extracted. A purification protocol was then developed (Table 1) to obtain homogeneous enzyme. Purification protocol All steps were performed at 4 °C and long-term storage occurred at )80 °C. The tubes for collecting the various fractions always contained 20 lL of a 10-m M stock PLP solution in order to increase the stability of kynureninase. The infected Sf9 cell culture (10 L) was harvested after 96 h by centrifugation at 5000 g for 7 min. Both the supernatant and the pellet (whole cells) were retained. Harvested insect cells were resuspended in cold buffer A consisting of 100 m M Tris/HCl buffer (pH 7.5) containing 0.25 M sucrose, 1 m M dithiothreitol, 0.5 m M EGTA, 10 l M PLP, 100 l M phenylmethanesulfonyl fluoride, 2 lgÆmL )1 apro- tinin plus 1 lgÆmL )1 pepstatin and leupeptin, and sonicated on ice. (All subsequent buffers contained the protease inhibitors, PLP, dithiothreitol and EGTA at the above concentrations.) The resultant fraction was then centrifuged at 12 000 g for 20 min. This procedure was repeated up to four times with retention of the supernatant. Both supern- atants were shown to contain all the activity. The superna- tant was brought to 20% (NH 4 ) 2 SO 4 saturation centrifuged at 10 000 g for 15 min and the pellet discarded. Then the (NH 4 ) 2 SO 4 was increased to 80% to precipitate kynuren- inase and centrifugation carried out as above. This pellet was redissolved in 3 mL buffer A, equilibrated with 50 mL 20 m M Tris/HCl at pH 8.6 and applied to a DEAE Blue Sepharose CL-6B affinity column (1.5 · 30 cm) that had been equilibrated with 10 m M Tris/HCl buffer at pH 8.6, at a flow rate of 1.5 mLÆmin )1 . The enzyme was eluted in the unbound fraction devoid of any PLP. Concentration and equilibration of fractions containing active kynureninase during this step and elsewhere were performed in an Amicon ultrafiltration unit incorporating a nitrocellulose membrane of exclusion limit 50 kDa. This system always retained the recombinant enzyme. Fractions containing active enzyme were pooled, equilibrated in 10 m M potas- sium phosphate buffer (pH 7.2), concentrated to 3.0 mL, and applied to a hydroxyapatite (Ultrogel) column (3.0 · 25 cm) equilibrated with 10 m M potassium phos- phate buffer at pH 7.2. The column with bound kynuren- inase was washed with 3 vol. equilibration buffer and then eluted with a stepwise gradient of sodium phosphate (10– 500 m M ) buffer at a flow rate of 1 mLÆmin )1 . Kynureninase was eluted at  160 m M . Active fractions were again pooled, equilibrated in 20 m M Tricine/NaOH, pH 8.8, concentrated to 4.0 mL and applied to a strong anion-exchange (Macro-Prep strong S support) column (1.5 · 30 cm) previously equilibrated with 20 m M Tricine/NaOH at pH 8.8 buffer at a flow rate of 2 mLÆmin )1 . Bound enzyme was washed with 3 column vol. equilibration buffer followed by stepwise elution with NaCl (10–500 m M ) in column equilibration buffer at a flow rate of 3.0 mLÆmin )1 .Theenzymewaselutedat60m M NaCl. A Macro-Prep strong Q support column (1.5 · 30 cm) was equilibrated with 20 m ML -His/HCl buffer at pH 6.0, and the pooled active fractions from the anion-exchange step were equilibrated (20 m ML -His/HCl, pH 6.0) and concentrated (4.0 mL) as previously. The concentrated fraction was applied to the column at a flow rate of 2.0 mLÆmin )1 , and bound kynureninase was washed with 3 column vol. equilibration buffer and eluted stepwise with KCl (0–400 m M )in20m ML -His/HCl buffer, pH 6.0, at a flow rate of 3.0 mLÆmin )1 . Kynureninase was eluted at  60 m M KCl. Samples containing kynureninase were pooled, concentrated (4.0 mL), and equilibrated in buffer (20 m M imidazole/HCl, pH 6.8) as described previously and applied to the strong anion-exchange column (1.5 · 30 cm) used earlier at a flow rate of 2.0 mLÆmin )1 . This column had been equilibrated with 20 m M imidazole/HCl buffer, pH 6.8. The enzyme was eluted in the unbound fraction and was pooled, concentrated, and equilibrated in assay buffer (10 m M Tris/HCl, pH 7.9), divided into aliquots, and stored at )80 °C until further use. The various purification steps were followed by SDS/PAGE (12% gels) [12]; where a Table 1. Fractional purification of recombinant human kynureninase from the supernatant of virus-infected insect (Sf9) cells. Specific details are outlined in the text. All activity assays were performed with 3-hydroxykynurenine as substrate and at saturating PLP. Step Total protein (mg) Total activity (nmolÆmin )1 ) Specific activity (nmolÆmin )1 Æmg )1 ) Fold purification % Yield 80% (NH 4 ) 2 SO 4 536 1460 2.7 1.00 100 Affi-Blue CL-6B 242 2183 9.0 3.30 150 Hydroxyapatite 96 3505 36.4 13.3 240 Strong anion @pH 8.9 29 3108 108 39.5 213 Strong cation @pH 6.0 16 2267 139 50.9 150 Strong anion @pH 6.0 8.0 1311 164 60.0 90 Scheme 1. 2070 H. A. Walsh and N. P. Botting (Eur. J. Biochem. 269) Ó FEBS 2002 tryptic mass fingerprint obtained by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS of a band of the expected molecular mass confirmed its identity as kynureninase. The protein concentrations were deter- mined with the Bradford assay [13]. Recombinant human kynureninase from the final anion-exchange step was assayed for purity using both SDS/PAGE and MALDI- TOF MS of the whole protein. Both purified and crude samples of recombinant kynureninase can be stored in the absence or presence of PLP for extended periods of time (> 12 months) at )80 °C without any loss of activity. At 4 °C the enzyme is stable for up to 2 weeks in the presence of PLP, and this is how crude enzyme solution was stored between successive steps. Activity assays Kynureninase activity of the enzyme was monitored spec- trofluorimetrically at 37 °C, with excitation of the product 3-hydroxyanthranilate at 330 nm and emission at 410 nm and 310 nm and 417 nm respectively for anthranilate, by the method of Shetty & Gaertner [7]. A Perkin–Elmer luminescence spectrometer (model LS50B) connected to a Grant circulating water bath was used for this purpose. The final reaction volume was 3.0 mL consisting of 25 nmol PLP (saturating), 10 m M potassium phosphate buffer at pH 7.9, substrate 3-hydroxykynurenine, and an appropriate volume of enzyme. Enzyme was always added last for all reactions including the inhibition studies. The amount of product formed was determined with reference to a standard curve of fluorescence intensity against 3-hydroxy- anthranilate concentration. The kinetic assays were per- formed using both crude and pure (> 95%) forms of the enzyme. Reproducibility of the experimental findings was confirmed with enzyme from different batches and varying degrees of purity in addition to replicates from within the same batch, and kinetic analyses showed no significant difference between the various extracts. A progress curve was constructed to confirm the linear relationship between product formation, protein concentration, and time. Lin- earity of the enzymatic reaction was determined over 5 min. To achieve temperature equilibration (37 °C), the assay mixture was incubated for at least 5 min before initiation of the reaction. Graphs were plotted using the CRICKETGRAPH and GraphPad PRISM 3 software packages, and the kinetic parameters K m and V max were obtained using non-linear regression. Lineweaver–Burk and Dixon [14] plots were used to characterize the type of inhibition. Hill analysis was also carried out to confirm the co-operativity. RESULTS Using the purification protocol described above, human recombinant kynureninase was purified > 60-fold from the supernatant fraction to yield active enzyme with a final specific activity of 164 nmolÆmin )1 Æ(mg protein) )1 .Thefull results of the purification are given in Table 1. It is not known why there is an initial increase in the activity during the purification (Table 1) given that the purification was performed at saturating PLP concentra- tions, however, there was no change in the electrophoretic mobility of the SDS/mercaptoethanol-treated enzyme from these crude extracts. It is possible that an inhibitor molecule has been removed during the purification procedure. Disc- gel electrophoresis in the absence of reducing agents SDS and mercaptoethanol to determine the native dimeric molecular mass resulted in the appearance of two bands at  52.5 and 95 kDa (gel image not shown), and this shows that the native protein exists mainly in the dimeric form. There was, however, a fair amount of tailing between the two bands which was probably due to the continuous association and disassociation of the respective subunits. Owing to the asynchronized viral infection cycle, lysis of a proportion of the transformed insect cells occurred, as was observed microscopically. This resulted in the presence of exogenous active kynureninase in the tissue culture medium. Hence an 80% (NH 4 ) 2 SO 4 precipitation was performed on the supernatant obtained from harvesting the whole cells. This fraction was purified separately, and the overall yield was significantly lower than the whole cell fraction but sufficient to warrant purification. The total pooled (super- natant + whole cells) enzyme activity from 10 L culture medium was  14 lmolÆmin )1 with a specific activity of 164 nmolÆmin )1 Æmg )1 (see Table 1 for fractional purifica- tion of the supernatant). The purified enzyme was shown to be purified to homogeneity (Fig. 1) by SDS/PAGE on a 12% gel with subsequent Coomassie Brilliant Blue staining. The molecular mass as determined by MALDI-TOF MS was 52.4 kDa, which is > 99.99% that of the predicted amino-acid sequence encoded by the cloned 1600 bp. A tryptic mass fingerprint obtained by MALDI-TOF MS also confirmed the identity of the protein as kynureninase. The UV absorption spectrum of the purified dimeric native protein at a concentration of 1.80 mgÆmL )1 in 10 m M Tris/ HCl buffer at pH 7.9 and 4 °C showed a peak at 432 nm, which was due to the presence of the PLP cofactor (data not shown). This scan was identical with that obtained by Kishore [15] for kynureninase from Pseudomonas marginalis. 2 Kinetic characterization of the recombinant human kynureninase revealed that the enzyme was specific for 3-hydroxykynurenine with an experimentally observed K m of 3.0 ± 0.10 l M for the racemic substrate ( D , L -3-hydroxy- kynurenine) [8,10]. Graphical analysis shows that the Fig. 1. Analysis of purified recombinant human kynureninase. SDS/ PAGE (12% gel) of the purified supernatant fraction showing kynu- reninase (30 lg) at 52.4 kDa in the presence of PLP. The gel was run as described by Laemmli [11]. Standards were Sigma prestained SDS molecular mass markers (SDS-7B) in sample buffer containing 4% SDS and 10% 2-mercaptoethanol. Ó FEBS 2002 Recombinant human kynureninase (Eur. J. Biochem. 269) 2071 enzyme is subjected to substrate regulation (graph not shown) and responds to both substrate and inhibitors in a sigmoidal fashion (Fig. 2). In contrast to previous reports, no substrate activity could be detected with L -kynurenine, using either a fluorimetric or UV spectroscopic assay. However, L -kynurenine was found to be a competitive inhibitor at low substrate concentrations (K i ¼ 20 l M ) and non-competitive at higher levels of substrate (K i ¢ ¼ 55 l M ) (Fig. 3). D -Kynurenine was also found to inhibit the enzyme (data not shown, K i ¼ 12 l M )asdida novel synthetic analogue, D , L -3,7-dihydroxydesaminoky- nurenine [16] (Scheme 2), with a K i of 100 n M .Thelatter two compounds were also found to be mixed inhibitors of the enzyme (data not shown). The pH optimum was determined as 8.25 and the activity of the enzyme was found to be strongly dependent on the ionic strength of the buffer. All assays, however, were performed at pH 7.9 because initial experiments with crude fractions were performed before the establishment of the pH-dependence curve. There was no significant difference in terms of reaction velocity between these two pH values. At pH 7.9, in 10 m M Tris/HCl buffer at 37 °C, velocity against substrate plots in the absence and presence (Fig. 2) of the inhibitor D , L -3,7-dihydroxydesaminokynurenine (100 n M ) were all distinctly sigmoidal, as was the percentage inhibition graph obtained with L -kynurenine (Fig. 4). A reciprocal plot of the data acquired for the D , L -3,7-dihydroxydesaminoky- nurenine-inhibited enzyme clearly reveals a highly co-operative enzyme throughout the whole substrate range, with negative co-operativity at low concentrations, which becomes positive as the substrate levels are increased (data not shown). Similar results were obtained by Hill analysis. DISCUSSION The results describe the first purification of human recom- binant kynureninase to homogeneity. The protein was fully Fig. 2. Kynureninase activity as a function of 3-hydroxykynurenine ([s]) in the absence (,) and presence [160 n M (h) and 5 l M (n)3,7- dihydroxydesaminokynurenine. Runin10l M Tris/HCl buffer (pH 7.9). Data are mean values of three replicate experiments, and the assay was performed as described in the text. Fig. 4. Inhibition of kynureninase activity by L -kynurenine as a function of 3-hydroxykynurenine concentration. Data obtained at 10 l M Tris/ HCl at pH 7.9, 37 °Cand15l M substrate concentration (d). The depicted kynureninase inhibition is expressed as the percentage of the inhibition with reference to the activity observed in the absence of inhibitor. Scheme 2. Fig. 3. Mixed inhibition of recombinant human kynureninase by L -kynurenine. Competitive inhibition (K i ¼ 20 l M ) observed at low concentrations of substrate which becomes mixed (K i ¢ ¼ 55 l M )at higher levels of substrate in 10 m M Tris/HCl buffer at pH 7.9 and 37 °C, K m ¼ 3.0 l M , specific activity of 164 nmolÆmin )1 Æ(mg pro- tein) )1 and n ¼ 3. Concentrations of L -kynurenine in l M were 0 (j), 16 (n), 32 (.), 64 (e), 128 (d) and 256 (h). 2072 H. A. Walsh and N. P. Botting (Eur. J. Biochem. 269) Ó FEBS 2002 characterized by electrophoresis (Fig. 1) 3 ,MALDI-TOFMS and UV absorption spectroscopy, and the data are consis- tent with previous reports [10,11] on the protein. The kinetic characterization revealed that the human recombinant kynureninase is specific for 3-hydroxykynurenine, with a K m of 3.0 ± 0.1 l M .ThisK m value is much lower than previously reported in this [11] and other laboratories [6] for the recombinant enzyme, and this is probably because our findings are for an enzyme displaying sigmoidal kinetics and thus the calculated ÔK m Õ is not the same as the Michaelis constant K m but rather a sigmoidal constant K s which incorporates an interaction factor(s) and hence is not the substrate concentration at 50% V max . The data are, however, consistent with values obtained for constitutive enzymes isolated from other species such as Saccharomyces cerevisiae (K m ¼ 3.0 l M ) [17] and the fungus 4 Penicillium roqueforti (K m ¼ 4.0 l M ) [7]. Soda & Tanizawa [18] reported a K m value of 1.67 l M for rat hepatic kynurenin- ase. It was also not possible to show any activity towards L -kynurenine, and, at the previously reported K m values of 400 l M or more, there was significant inhibition of the enzyme. At a concentration of 250 l ML -kynurenine in the presence of 625 n MD , L -3-hydroxykynurenine, there was nearly 80% inhibition (Fig. 4). This is a major difference of human kynureninase from other mammalian enzymes, such as rat hepatic kynureninase, and may imply that previous reports of weak activity with L -kynurenine in crude cell homogenates may be the result of additional adventitious enzyme activity. Certainly the preference for 3-hydroxyky- nurenine must be taken into account in inhibitor design. Rat hepatic kynureninase, on the other hand, showed activity towards L -kynurenine, with a K m of 500 l M for the partially purified (> 80%) enzyme. Differential substrate specificity for kynureninase from brain and liver in mice has been demonstrated by Chiarugi et al.[19]in vivo, and this raises the possibility of the existence of two isoforms of the enzyme as discussed by Toma et al.[8]. The pH optimum of 8.25 is consistent with previously reported experimentally determined [6] values, and the optimum activity of the enzyme using this potassium phosphate buffer system showed a strong dependence on ionic strength. Molarity increases above 10–20 m M result in a significant fall in activity, which progressively worsens as the ionic strength is increased. On the basis of the sigmoidal velocity plots obtained in the absence and presence of D , L -3,7-dihydroxydesaminoky- nurenine (Fig. 2), the enzyme appears to be subjected to co-operative modulation by the substrate 3-hydroxykynur- enine. The mixed inhibition depicted by the Lineweaver– Burk plot in Fig. 3 corroborates this with its reduced V max and increased K m . The shape of the plot is consistent with binding of the inhibitor to both the free enzyme (E) and the enzyme–substrate complex (ES) [15] and hence it can be inferred that an additional ligand-binding site must be present on the human enzyme. When the lines intersect above the x-axis then K i < K i ¢, and when the lines intersect below the x-axis then K i > K 0 i (in both instances the lines have to intersect to the left of the y-axis). The data obtained for L -kynurenine gave K i ¼ 20 l M and K i ¢ ¼ 55 l M , while the D -isomer (data not shown) showed very similar behavior. CONCLUSIONS The work described shows that appreciable quantities of active recombinant human kynureninase can be obtained using a baculovirus/insect cell system followed by a straightforward purification protocol. This provides a relatively simple and economical method of producing active enzyme for use in mechanistic and structural studies. Characterization of recombinant human kynureninase shows that it is similar to the rat liver enzyme [4] in terms of molecular mass, pH optimum, K m and sensitivity to analogue inhibitors, but it also has some important differences. The human enzyme seems to be completely specific for 3-hydroxykynurenine with no significant activity with kynurenine, as reported previously. This may be important in inhibitor design. Also the enzyme appears to be subjected to substrate modulation, exhibiting sigmoidal kinetics. This behaviour could be important in regulation of enzyme activity in vivo and consequent channeling of substrate 3-hydroxykynurenine down the tryptophan– kynurenine metabolic pathway. The purification of recombinant human kynureninase to homogeneity has allowed crystallization trials to commence in our laboratory for elucidation of the X-ray crystal structure of the protein. The information obtained should provide invaluable knowledge on the active site and also pave the way for co-crystallization of enzyme–substrate and/or enzyme–inhibitor complexes. These should allow further mechanistic investigation of the catalytic reaction and hence facilitate subsequent design and synthesis of effective inhibitors in an attempt to combat the deleterious effects of the many serious neurodegenerative disorders. ACKNOWLEDGEMENTS A fellowship to H. A. W. from the Wellcome Trust provided the funds for this study. Dr C. H. Botting is acknowledged for helpful discussions, performing the MALDI-TOF MS, and valuable compu- ting assistance. REFERENCES 1. Baran, H., Cairns, N. & Lubec, B. (1996) Increased kynurenic acid levels and decreased brain kynurenine aminotransferase I in patients with Downs syndrome. Life Sci. 58, 1891–1895. 2. Botting, N.P. (1993) Chemistry and neurochemistry of the kynurenine pathway of tryptophan metabolism. Chem. Soc. Rev. 45, 309–315. 3. Stone, T.W. (2000) Development and therapeutic potential of kynurenic acid and kynurenine derivatives for neuroprotection. Tresnds Pharmacol. Sci. Rev. 21, 149–154. 4. Takeuchi, F., Otsuka, H. & Shibata, Y. (1980) Purification and properties of kynureninase from rat liver. J. Biochem. (Tokyo) 88, 987–994. Scheme 3. Ó FEBS 2002 Recombinant human kynureninase (Eur. J. Biochem. 269) 2073 5. Nishizuka, Y. & Hayaishi, O. (1963) Studies on the biosynthesis of nicotinamide adenine dinucleotide. J. Biochem. (Tokyo) 238, 3368–3377. 6. Mellilo, G., Musso, T., Sica, A., Taylor, L., Cox, G.W. & Varesio, L. (1995) A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med. 182, 1683–1693. 7. Shetty, A.S. & Gaertner, F.H. (1973) Distinct kynureninase and hydroxykynureninase activities in microorganisms: occurrence and properties of a single physiologically discrete enzyme in yeast. J. Bacteriol. 113, 1127–1133. 8. 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(1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 14. Dixon, M. & Webb, E.C. (1964) The Enzymes, 2nd edn, pp. 116– 145. Academic Press, New York. 15. Kishore, G.M. (1984) Mechanism-based inactivation of bacterial kynureninase by b-substituted amino acids. J. Biol. Chem. 259, 259–264. 16. Walsh, H.A., Leslie, P.L., O’Shea, K. & Botting, N.P. (2002) 2-Amino-4-[3¢-hydroxyphenyl]-4-hydroxybutanoic acid; a potent inhibitor of rat and recombinant human kynureninase. Biorg. Med. Chem. Lett. 12, 361–363. 5 17. Schott, H H. & Krause, U. (1979) Purification and charac- terization of 3-hydroxykynureinase from yeast. Z Physiol. Chem. 360, 481–488. 6 18. Soda, K. & Tanizawa, K. (1979) Kynureninases: enzymological properties and regulation mechanism. Adv. Enzymol. Relat. Areas Mol. Biol. 49, 1–40. 19. Chiarugi, A., Carpanedo, R. & Moroni, F. (1996) Kynurenine disposition in blood and brain of mice: effects of selective inhibitors of kynurenine hydroxylase and of kynureninase. J. Neurochem. 67, 692–698. 2074 H. A. Walsh and N. P. Botting (Eur. J. Biochem. 269) Ó FEBS 2002 . Purification and biochemical characterization of some of the properties of recombinant human kynureninase Harold A. Walsh and Nigel P. Botting School of. of human recom- binant kynureninase to homogeneity. The protein was fully Fig. 2. Kynureninase activity as a function of 3-hydroxykynurenine ([s]) in the