catalyst is specifically a hydroxykynureninase

Transcription

1 JOURNAL OF BACrERIOLOGY, Apr. 1975, p Copyright i 1975 American Society for Microbiology Vol. 122, No. 1 Printed in U.S.A. Kynureninase-Type Enymes of Penicillium roqueforti, Aspergillus niger, Rhiopus stolonifer, and Pseudomonas fluorescens: Further Evidence for Distinct Kynureninase and Hydroxykynureninase Activities A. S. SHETITYI AND F. H. GAERTNER* Department of Biology, Florida Agricultural and Mechanical University, Tallahassee, Florida 32307, and the University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830* Received for publication 13 December 1974 The kynureninase-type enymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalye the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhiopus stolonifer was found to contain a single, constitutive enyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 x 106 and 2.5 x 10-' M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 x 10-' to 14.3 x 10-5 M, as well as a constitutive enyme with Km for the two substrates of -4 x 10-6 M and 10-i M. The bacterium Pseudomonas fluorescens has a single, inducible enyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 x 10-i and 7 x 1O-5 M. In addition, significant differences in maximal velocities (V.max) 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 VImax 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 enyme. In Neurospora crassa L-tryptophan may be degraded and excreted as anthranilate or it may be metabolied to 3-hydroxyanthranilate and used in a pathway leading to nicotinamide adenine dinucleotide (NAD) biosynthesis. Previously, it was thought that a single, inducible enyme, kynureninase (L-kynurenine hydrolase, EC ), was responsible for the formation of both anthranilate metabolites (3). Hence, the kynureninase of N. crassa has even been presented as one of the few examples of an inducible biosynthetic enyme (11). More recently, however, Gaertner et al. (1) have shown that a distinct, constitutive enyme is responsible for the synthesis of 3-hydroxyanthranilate in N. crassa. Unlike the inducible activity, the con- one has a low Km for L-3-hydroxyky- stitutive nurenine. For this reason and because the ' Present address: Department of Biology, Florida A & M University, Tallahassee, Fla constitutive enyme predominates in cells cultured in the absence of a tryptophan supplement, it was proposed that the constitutive catalyst is specifically a hydroxykynureninase (1). Although other such constitutive hydroxykynureninase activities have recently been shown to occur in yeast (6) and in mammalian liver (4), the generality of these results and the conclusions made concerning the physiological significance of both the inducible and the constitutive enymes would be significantly strengthened with a few well-chosen additional examples. Specifically, in such an analysis one could determine if it is generally true that (i) organisms catalying the biosynthesis of NAD from tryptophan contain a constitutive hydroxykynureninase, (ii) constitutive kynureninase-type enymes have low Km for L-3-hydroxykynurenine (The term kynureninase-type enyme as used here signifies any enyme with the 235

2 236 SHETTY AND GAERTNER J. BACTrERIOL. capacity to hydrolye L-kynurenine or its analogues, such as L-3-hydroxykynurenine, or N- formylkynurenine to anthranilate or its analogue, such as L-3-hydroxyanthranilate or N- formylanthranilate. In practice the kynureninase-type enymes are demonstrated by their ability to hydrolye L-kynurenine to anthranilate.), (iii) inducible kynureninase-type enymes have relatively low Km for L-kynurenine, (iv) organisms carrying an inducible kynureninase excrete anthranilate in response to L-tryptophan, and (v) the inducible enyme can substitute for the constitutive enyme in those organisms where both enymes are normally present. For the above analysis we have selected four microorganisms for further study: Aspergillus niger, Penicillium roqueforti, Rhiopus stolonifer, and Pseudomonas fluorescens. The first three are fungi which presumably catalye the synthesis of NAD from tryptophan. In an initial study it was determined that the first two, both ascomycetes; contain an inducible kynureninase-type enyme and excrete anthranilate in response to L-tryptophan. The third organism, a phycomycete, showed no such response. The fourth, a bacterium, was chosen because it is known to contain an inducible kynureninasetype enyme (2) but is incapable of synthesiing NAD from tryptophan, presumably utiliing the aspartate plus glyceraldehyde 3-phosphate pathway instead (8). In the present investigation, the kynureninase-type enymes of these four organisms are isolated and their kinetic properties with the substrates L-kynurenine and L-3-hydroxykynurenine are determined. MATERIALS AND METHODS Organisms. The bacterial and fungal strains used in this study are P. fluorescens (ATCC 11250), R. stolonifer (ATCC 14037), P. roqueforti (ATCC 10110), and A. niger (ATCC 16888). Growth of organisms. P. fluorescens was grown in minimal medium of Vogel and Bonner (10), modified to contain only one-half the MgSO4. The cells were grown with and without 400 og of L-tryptophan per ml, with 5 g of dextrose per liter as the carbon source. Cells from 5-ml nutrient agar slants were inoculated to 1-liter Erlenmeyer flasks containing 500 ml of appropriate sterile media. The flasks were incubated for 2 days at 25 C in a model G25 incubator-shaker (New Brunswick Scientific Co., Inc.) at 150 rpm. Each flask was used to inoculate one 20-liter Nalgene carboy containing 15 liters of medium. After 24 h of growth under forced aeration at 25 C, the cells were harvested with a Sharples centrifuge. Each carboy yielded about 35 g (wet weight) of cells. Each of the fungi was grown in Vogel minimal medium (9) containing 1 g of yeast extract and 20 g of sucrose per liter. Spores were washed from 5-ml potato dextrose agar slants with 6 ml of minimal medium. Six 1-liter Erlenmeyer flasks containing 500 ml of sterile medium were each inoculated with 1 ml of the spore suspension. The flasks were incubated at 25 C for 2 days, as described above, in an incubator shaker. Each flask was used to inoculate one of six 20-liter Nalgene carboys containing 15 liters of medium. Three of the carboys were supplemented with 200 Mg of L-tryptophan per ml. The mycelium was harvested in cheesecloth after 2.5 days of growth under rapid forced aeration at 25 C. The total yield of lyophilied mycelium from three carboys was about 75 g. For initial screening of organisms, crude extracts were prepared by grinding the filtered and partially dried fungi in approximately 10 volumes of 0.1 M potassium phosphate, ph 7.0, with a close-fitting, hand-operated, ground-glass homogenier; the bacterium was prepared by passing the cells suspended in the same 0.1 M phosphate buffer through a French press set at approximately 20,000 lb/in2. In each case after the disrupted preparation was centrifuged, assays were performed on the supernatant portion. Extraction and purification of enyme. The bacterial cells (P. fluorescens, 35 g [wet wt]) were suspended in 4 volumes of 0.05 M potassium phosphate (ph 7) and were broken by one passage through a French press at 18,000 to 20,000 lb/in2. The viscous homogenate (122 ml) was treated with about 0.1 mg of Worthington deoxyribonuclease and was warmed to room temperature for a few minutes to digest the nucleic acids. After the extract was centrifuged at 10,000 x g for 30 min, the supernatant crude extract was subjected to protamine sulfate precipitation, precipitation at 60% of saturation with ammonium sulfate, Sephadex G-25 gel filtration, and diethylaminoethyl (DEAE)-cellulose chromatography. These were carried out as described previously (1), with only one modification: the DEAE-cellulose chromatography was performed with two successive ph 6.5 potassium phosphate gradients of 0.01 to 0.15 and 0.15 to 0.5 M. Both gradients were 2 liters each, and fractions of about 20 ml were collected every 28.8 min. The mycelia from the two ascomycetes were lyophilied in a Virtis lyophilier. The dried mycelia were ground to a fine powder with a Wiley mill, and extracts from 75 g of each powder were prepared (1). Other purification procedures were the same as those described for P. fluorescens. The phycomycete, R. stolonifer, was handled in the same manner as the other fungi except that extracts were prepared from 2.5 g of lyophilied mycelium and DEAE-cellulose chromatography was not performed. Assays. The kynureninase (L-kynurenine to anthranilate) and the hydroxykynureninase (L-3- hydroxykynurenine to 3-hydroxyanthranilate) activities were determined using an Aminco Bowman spectrophotofluorometer as described in earlier reports (1, 6). Activities are expressed in international units at 25 C. Protein concentration was estimated at 280 nm in a Gilford model 2400 spectrophotometer.

3 VOL. 122, 1975 RESULTS Initial experiments with the organisms chosen for study in this investigation revealed the following facts: when A. niger, P. roqueforti, and P. fluorescens were grown in the appropriate media supplemented with L-tryptophan, as described in Materials and Methods, a fluorescence characteristic of anthranilate appeared in the medium indicating a concentration of between 10- I and 6 x 10-4 M. Chromatographic analysis of the medium, performed as described by Shetty and Gaertner (6), confirmed in each case the identity of the fluorescent material as anthranilate. (The chromatographic solvent used [reference 6], butanol-propanol-water- NH,OH [25:50:25:0.25 ], causes the spontaneous oxidation of 3-hydroxyanthranilate to cinnabarinic acid [R1, 0.15] but does not alter anthranilate [R, 0.5]. Inclusion of 0.25 ml of - 2-mercaptoethanol in 100 ml of solvent prevents the oxidation and provides a spot with an Rf 0.37 [F. H. Gaertner, unpublished data].) On the other hand, when each organism was grown in medium unsupplemented with L-tryptophan, the medium remained free of this characteristic fluorescence. These results indicated that each of the organisms contains an inducible kynureninase-type enyme. This expectation was confirmed by assaying crude extracts for such activity. In all three examples the kynurenineto-anthranilate activity was high in cells grown in media supplemented with L-tryptophan and was low or undetectable in cells from unsupplemented media. Similar experiments performed with R. stolonifer revealed for the first time that at least one example of the phycomycetes does not excrete anthranilate in response to L-tryptophan in the medium, but instead excretes 3-hydroxyanthranilate. Moreover, the specific activity of its kynureninase-type enyme is unchanged in the presence of L-tryptophan. To determine the nature of the kynureninasetype enymes contained in the selected organisms, the enymes in each were purified sufficiently to reveal individual activities. After DEAE-cellulose chromatography, overall recovery determined by summation of the kynureninase activity in the peak fractions was between 20 and 50%, and overall purification from the crude extracts was typically 30- to 50-fold in peak fractions. DEAE-cellulose chromatography of ammonium sulfate fractions from A. niger, P. roqueforti, and P. fluorescens substantiated the occurrence of a single inducible kynureninasetype activity in each (Fig. 1B, 2B, and 3) and indicated the presence of a second, much lower KYNURENINASE-TYPE ENZYMES 237 level, constitutive activity in both A. niger and P. roqueforti (Fig. 1A and 2A). P. fluorescens, on the other hand, lacked such a constitutive enyme since no kynureninase-type activity could be detected in crude extracts, or in partially purified fractions from cells unsupplemented with tryptophan (data not shown). Similarly, the inducible activity of A. niger was undetectable in cells unsupplemented with L- tryptophan, but a small peak of activity was present, located approximately 14 fractions later in the chromatographic separation (Fig. 1A). Although this second peak of activity was not completely resolved from the high level of inducible enyme in the case of the supplemented cells (Fig. 1B), the amount of activity at the position of this second peak (fractions 90 to 100) was the same in the chromatograms of both the supplemented and unsupplemented preparations, indicating that this second peak of activity is not induced by L-tryptophan. Unlike A. niger, the inducible activity did not fall to an undetectable level in the unsupplemented cells of P. roqueforti. However, as in A. niger, a second small peak of activity was observed in the unsupplemented case located approximately 12 fractions later in the column effluent (Fig. 2A and 2B). The constitutivity of the enyme in the second peak is suggested by the fact that only one peak of inducible activity is present in the chromatogram of the extract from the cells supplemented with L-tryptophan (Fig. 2B). Finally, in the case of R. stolonifer the specific activity of the kynureninase-type enyme was unaffected by the presence of L-tryptophan in the medium (Table 1). Hence it was apparent that this organism contained only a constitutive enyme. The experiments described in the following section also support the above points. Kinetic analysis of the isolated inducible and constitutive activities verified the fact that they were catalyed by distinct enymes. The inducible enymes from each organism were found to have low Km for L-kynurenine; in contrast, the constitutive activities had low Km for L-3- hydroxykynurenine (Fig. 4; Table 2). Of the enymes tested, the constitutive activity of R. stolonifer proved to have the highest degree of specificity for L-3-hydroxykynurenine, whereas the inducible enyme of P. fluorescens showed the highest degree of specificity for L-kynurenine. In both these examples Km as well as Vmax parameters contributed to the specificities. In all other examples where differences occur, only the Km differs significantly for the two substrates. Of interest and perhaps of physiological

4 238 SHETTY AND GAERTNER J. BAC'rIOL. E.E-: "I LU y LUJ 0 Lr CI I) 0 I a. Downloaded from ~~~~~~~~~~~0.4 2-,,o'' ;!- 0, FRACTION NO, FIG. 1. DEAE-cellulose chromatography of extracts from A niger. The cells were grown in minimal medium (A) and in medium supplemented with 200 ug of L-tryptophan per ml (B). Enyme purification was carried out as described in Materials and Methods. The inset in the unsupplemented case shows the active fractions on a 20-fold greater scale than that used elsewhere in the figure. Symbols: A2M (broken line), enyme activity in international units x10-8 (-4), and potassium phosphate concentration (solid line). significance is the fact that the inducible enymes of A. niger and P. roqueforti, while having distinctly low Km for L-kynurenine as opposed to their constitutive enymes, had Km and Vmax values which were as favorable for L-3-hydroxykynurenine as for L-kynurenine. Thus, these enymes are the least specific of the kynureninase-type enymes examined. on November 6, 2018 by guest

5 VOL. 122, 1975a KYNURENINASE-TYPE ENZYMES 239 U-).?.2 cn y 0 Li LI C,) 0 I a- Downloaded from FRACTION NO. FIG. 2. DEAE-cellulose chromatography of extracts from P. roqueforti. The cells were grown in minimal medium (A), and in medium supplemented with 200 lag of L-tryptophan per ml (B). Enyme purification was carried out as described in Materials and Methods. The inset in the unsupplemented case shows the active fractions on a 250-fold greater scale than that used elsewhere in the figure. Symbols: A 2.. (broken line), enyme activity in international units x 10-(I - ), and potassium phosphate concentration (solid line). Because the inducible activity did not fall to an undetectable level in P. roqueforti and because the second peak of activity was unusually low, the nature of the remaining inducible enyme and the constitutivity of the enyme in the second peak required further analysis. Determination of the Km and relative Vm.. with L-kynurenine and L-3-hydroxykynurenine showed that the inducible activity remaining in unsupplemented cells and the activity in the tailing fractions of the induced peak were the same as that found for the fraction with the highest activity from the induced peak. Thus, the only activity showing a low Km for L-3- hydroxykynurenine was that from the second small peak. Since no indication of this activity was found in the tailing fractions of the main peak, it can be concluded that this enyme is on November 6, 2018 by guest

6 240 SHETTY AND GAERTNER J. BACTERIOL. 1- \ LU U) E Lu 'D y FRACTION NO. FIG. 3. DEAE-cellulose chromatography of an extract from Pseudomonas fluorescens. The cells were grown in medium supplemented with 400 Ag of L-tryptophan per ml. No activity was observed in cells unsupplemented with L-tryptophan. Enyme purification was carried out as described in Materials and Methods. Symbols: A280 (broken line), enyme activity in international units x10' (@-), and potassium phosphate concentration (solid line). TABLE 1. Purification of kynureninase-type enyme from Rhiopus stolonifer Kynuren- Total Total inase Overall Overall Fraction volume activity sp act yield enrich- (ml) (IU x 10-3) A280 (IU x 10-/ (%) ment A280) Supplemented with L-tryptophan Crude extract Protamine sulfate supernatant Ammonium sulfate precipitate (0-60%) Sephadex G-25 eluate Unsupplemented with L-tryptophan Crude extract Protamine sulfate supernatant Ammonium sulfate precipitate (0-60%) Sephadex G-25 eluate not significantly induced by L-tryptophan. A similar analysis performed on the tailing fractions of the induced peak of kynureninase-type activity from A. niger verified the fact that these fractions contained the constitutive activity in that they had a low Km for L-3-hydroxykynurenine (data not shown). DISCUSSION Previous observations on the kynureninasetype activities of N. crassa (1), Saccharomyces cerevisiae (6), and Mus musculus (4) suggested that the constitutive enymes contained in each organism were specifically hydroxykynureninases, whereas the inducible enyme carried by N. crassa was a relatively specific kynureninase. To substantiate these conclusions and to examine their significance with respect to other cell types, three definitive categories of microorganisms were selected for analysis. For example, based on prior knowledge of the mode of NAD synthesis and the content of kynureninase-type cr (5 LUJ 0- r u) 0 I: a-

7 VOL. 122, A KYNURENINASE-TYPE ENZYMES c N 0.06 Un a) w 0 E 0 C o C C Downloaded from KYNURENINE OR HYDROXYKYNURENINE (mm) FIG. 4. Reaction rates of kynureninase-type enymes as a function of L-kynurenine (O-4) and L-3-hydroxykynurenine (O-O) concentrations. Reactions were performed as described in Materials and Methods except that the substrate concentrations were varied as indicated. The enyme preparations used in these studies were DEAE-cellulose peak fractions from all organisms except R. stolonifer, in which case a Sephadex G-25 eluate of an ammonium sulfate fraction as described in Materials and Methods was used. Linear data are plotted on semilogarithmic graphs in order to provide a comprehensive comparison of the results. K. values obtained from double reciprocal plots (not shown) are indicated by arrows. (A) A. niger minus L-tryptophan, fraction 93 of Fig. 1A; (B) A. niger plus L-tryptophan, fraction 71 of Fig. IB; (C) P. roqueforti minus L-tryptophan, fractions of Fig. 2A; (D) P. roqueforti plus L-tryptophan, fraction 51 of Fig. 2B; (E) R. stolonifer minus L-tryptophan, Sephadex G-25 eluate, Table 3; (F) P. fluorescens plus L-tryptophan, fraction 103 of Fig. 3. on November 6, 2018 by guest

8 242 SHETTY AND GAERTNER J. BACTrERIOL. TABLE 2. Kinetic properties of kynureninase-type enymesa Inducible enyme Constitutive enyme Microorganism Substrate V,,a, (lu/ml Vmax (IU/Ml K.6 (M) of assay) K.6 (M) (of assay) Pseudomonas fluorescensb Kynurenine 7.14 x x 10-2 NPc NPc Hydroxykynurenine 5.00 x 10-' 3.03 x 10-2 NPe NPc Aspergillusnigee Kynurenine 8.0 x 10-' 1.79 x x 10-' 2.44 x 10-4 Hydroxykynurenine 1.43 x 10-' 1.20 x x x 10-' Penicillium roqueforti6 Kynurenine 7.14 x x 10-l 1.00 x x 10-' Hydroxykynurenine 5.88 x x x 10-' 7.41 x 10-' Rhiopus stoloniferd Kynurenine NPc NPc 2.50 x x 10-' Hydroxykynurenine NPc NPc 6.67 x x 10-' a Enyme assays were performed kinetically as described previously (6), and the data are from double reciprocal plots of results presented in Fig. 4. b DEAE-cellulose peak fraction was used as enyme (cf. Fig. 1-3). c NP, Not present. d Sephadex G-25 eluate was used as enyme (cf. Table 1). activity, the phycomycete R. stolonifer was selected as a representative of one category. Since Rhiopus was found in initial experiments to contain only a constitutive kynureninasetype activity and to excrete 3-hydroxyanthranilate instead of anthranilate, it could be predicted that its constitutive enyme would have a low Km for L-3-hydroxykynurenine and a high one for L-kynurenine. That this was found to be the case establishes R. stolonifer as a fourth example of an organism containing only a hydroxykynureninase. Thus, S. cerevisiae (6), M. musculus (4), Rattus novigicus (A. De Antoni, C. Costa, and G. Allegri, ISTRY-Abstr. First Int. Meet. Tryptophan Metabol.: Biochem. Pathol. Reg., 1974), and R. stolonifer each contain a constitutive hydroxykynurenine hydrolase as the enyme required for catalying the fourth step in the biosynthesis of NAD from L-tryptophan. In a second category two organisms were selected, A. niger and P. roqueforti. Although these ascomycetes also presumably synthesie NAD from L-tryptophan, initial studies showed that like N. crassa each contained induced levels of a kynureninase-type enyme and excreted anthranilate when cultured in media supplemented with L-tryptophan. Therefore, based on the previous results with N. crassa it could be predicted (i) that the inducible activity in each organism would have a relatively low Km for L-kynurenine, (ii) that both organisms would be found to contain also a constitutive kynureninase-type enyme, and (iii) that this constitutive activity would prove to have a low Km for L-3-hydroxykynurenine. Except that the constitutive activity of P. roqueforti was unusually low, these predictions were substantiated. Moreover, the fact that in Aspergillus the inducible activity fell to an undetectable level in cultures unsupplemented with L-tryptophan supports strongly the contention that this constitutive enyme is the only kynureninase-type enyme responsible for the synthesis of NAD in these cells. Whether the low level of constitutive enyme in P. roqueforti is an artifact of preparation or is physiologically significant remains to be determined. However, the facts that the inducible activity remains at a relatively high level in cells cultured in the absence of L-tryptophan and has a Km as low for L-kynurenine as for L-3-hydroxykynurenine leaves open the possibility that the inducible activity in these cells could be involved in the synthesis of NAD. In a third category we chose P. fluorescens as an example of an organism which contains an inducible kynureninase-type activity and which lacks the ability to synthesie NAD from L-tryptophan, synthesiing it instead from aspartate and glyceraldehyde 3-phosphate. Since there is apparently no biosynthetic role for the kynureninase-type enyme contained in this bacterium, it could be predicted (i) that the inducible activity would have a high Km for L-3-hydroxykynurenine and a low one for L-kynurenine, and (ii) that the organism would lack a constitutive activity. Since P. fluorescens is devoid of any detectable kynureninase-type activity when the cells are cultured in the absence of L-tryptophan and since the inducible kynureninase enyme it contains not only has a high Km for L-3-hydroxykynurenine but a significantly lower Vmax for this metabolite than for L-kynurenine, our predictions were again verified. Therefore, P. fluorescens is established as the first documented example of an organism containing only a specific inducible kynureninase Ḟrom these results the following general expectations for the kynureninase-type enymes

9 VOL. 122, 197'a KYNURENINASE-TYPE ENZYMES 243 were fulfilled. (i) The organisms synthesiing NAD from L-tryptophan were found to contain a constitutive kynureninase-type enyme with a low Km for L-3-hydroxykynurenine (-5 x 10-6 M). (ii) The organisms which excrete anthranilate in the medium in response to L-tryptophan were found to contain an inducible kynureninase-type enyme with a low Km for L-kynurenine (- 7 x 10-5 M). (iii) The seven constitutive kynureninase-type enymes thus far described were found to have low Km for L-3-hydroxykynurenine and could be characteried as specific hydroxykynureninases. (iv) The four inducible kynureninase-type enymes described were all found to have low Km for L-kynurenine and could be characteried as kynureninases. It is not yet clear to what extent the inducible kynureninases may substitute for a missing or defective constitutive hydroxykynureninase in the biosynthesis of NAD. Mutants lacking the constitutive hydroxykynureninase have not been isolated, but we are attempting to isolate them in N. crassa. The unusually low level of constitutive hydroxykynureninase and concurrently unusually high level of inducible kynureninase in P. roqueforti may represent a natural example of such an adaptation. Furthermore, it is our hypothesis that the tryptophan-to-nad pathway evolved from an aerobic catabolic pathway which in more advanced organisms came to replace the anaerobic aspartate plus glyceraldehyde 3-phosphate pathway (F. H. Gaertner and A. S. Shetty, manuscript in preparation). As will be shown, this hypothesis requires the existence at some point of organisms containing only an inducible kynureninase-type enyme but with the capacity to synthesie NAD from L-tryptophan. Finally, several points raised recently in the literature need clarification. Two inducible forms of kynureninase in N. crassa were described previously by Turner and Drucker (7). These were referred to as Kyn I and Kyn II. Since both activities were elevated by the addition of L-tryptophan to the medium, neither can be confused with the constitutive hydroxykynureninase, which is unaffected by such additions. Although we have been unable to demonstrate two forms of inducible activity in N. crassa, or in any other organisms we have investigated, we do not exclude the possibility that a third kynureninase-type enyme exists in N. crassa. However, it is difficult to understand what the physiological significance of such an enyme would be. Therefore, pending establishment of the existence of the two inducible enymes, we recommend that they be referred to as Kyn I and Kyn II, as suggested by Turner and Drucker (7), and that the constitutive kynureninase-type enyme be referred to as either hydroxykynureninase, L-3-hydroxykynurenine hydrolase, constitutive kynureninasetype enyme, or if necessary, Kyn Ill. More recently Schlitt et al. (5) confirmed the existence of the constitutive enyme in N. crassa. Unfortunately, they failed to do the kinetic studies necessary to establish the specificity of the enyme as a hydroxykynureninase. Rather, they referred to the activity as Kyn II and questioned the term hydroxykynureninase based only on the observations that they could not see a difference in maximal activity for L-3-hydroxykynurenine or L-kynurenine, and that the enyme was not bound to mitochondrial membrane. Since the difference in maximal activity for L-3-hydroxykynurenine or L- kynurenine is either small or insignificant in all of the hydroxykynureninases thus far isolated, except that of R. stolonifer, and since L-kynurenine hydroxylase is the only tryptophan-to- NAD enyme demonstrated in any organism to be bound to the mitochondrial membrane, we consider their arguments to be without basis, and their reference to the constitutive enyme as Kyn II to be inappropriate. The fact that none of the kynureninase-type enymes shows a total specificity for either L-kynurenine or L-3- hydroxykynurenine may pose some problems in nomenclature. However, irrespective of the eventual names proposed for these enymes, the conclusion that functionally discrete L-kynurenine and L-3-hydroxykynurenine hydrolases exist in various organisms seems inescapable. ACKNOWLEDGMENTS This research was sponsored by the U.S. Atomic Energy Commission under contact with the Union Carbide Corporation. It was supported by a grant to the University of Tennessee by the Carnegie Corporation of New York and by Minority School Biomedical Support grant RR-0811 from the NIH to the Florida A & M University. We wish to thank K. W. Cole for growing the fungi. LITERATURE CITED 1. Gaertner, F. H., K. W. Cole, and G. R. Welch Evidence for distinct kynureninase and hydroxykynureninase activities in Neurospora crassa. J. Bacteriol. 108: Hayaishi, O., and R. Y. Stanier The kynureninase of Pseudomonas fluorescens. J. Biol. Chem. 195: Jakoby, W. B., and D. M. Bonner Kynureninase from Neurospora: purification and properties. J. Biol. Chem. 205: McDermott, C. E., D. A. Casciano, and F. H. Gaertner Isolation and characteriation of an hydroxykynureninase from homogenates of adult mouse liver.

10 244 SHETTY AND GAERTNER J. BACTERIOL. Biochem. Biophys. Res. Commun. 51: Schlitt, S. C., G. Lester, and P. J. Russell Isolation and characteriation of low-kynureninase mutants of Neurospora crassa. J. Bacteriol. 117: Shetty, A. S., and F. H. Gaertner Distinct kynureninase and hydroxykynureninase activities in microorganisms: occurrence and properties of a single physiologically discrete enyme in yeast. J. Bacteriol. 113: Turner, J. R., and H. Drucker Kynureninase from Neurospora: occurrence of two activities. Biochem. Biophys. Res. Commun. 42: Uhlik, D. J., and C. S. Gowans Synthesis of nicotinic acid in Chlamydomonas eugametos. Int. J. Biochem. 5: Vogel, H. J A convenient growth medium for Neurospora (medium N). Microbial Genet. Bull. 13: Vogel, H. J., and D. M. Bonner Acetylornithinase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218: Wainwright, S. D Control mechanisms and protein synthesis, p Columbia University Press, New York.