A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolas

Gaertner, F. A.

Fungal Genetics Reports

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Kansas State University

Fungal Genetics Reports Volume 28 Article 7 A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase F. A. Gaertner Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Gaertner, F. A. (1981) "A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase," Fungal Genetics Reports: Vol. 28, Article 7. https://doi.org/10.4148/1941-4765.1652 This Research Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact cads@k-state.edu. A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase Abstract A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol28/iss1/7 Gaertner, F. A. The nt lesion must involve tryptophan pyrrolase(tryptophan-2, 3-dioxygenase, EC 1.13.1.12). However,A response to a letter from David Perkins we still have no direct evidence that this is thecase since tryptophan oxygenase has not been detectedconcerning the question of whether nt in N. crassa  or for that matter in any other moldthat we have tested: Mucor, Rhizopus, Aspergillus,mutants involve tryptophan pyrrolase. Penicillium, and Saccharomyces (Gaertner and Salvoni,unpublished results). We have a very sensitive fluo-rometric assay (1973 Arch. Biochem. Biophys. 156:658)  that works well for liver, bacterial and Drosophila tryptophan oxygenases. Our initial report of suc-cess in assaying the enzyme in Neurospora crassa slime still holds. Unfortunately, however, this onlyamounts to a whole cell assay and so far has not been useful for wild type N. crassa. Hence, we have onlybeen able to assay for the presence or absence of tryptophan oxygenase in nt mutants. The following storydescribes what we do know about nt mutants (unpublished data).In an attempt to isolate mutants of 3-deoxy-D-arabinoheptulosanate 7-phosphate (DAHP) synthase (trp)in N. crassa , and to test the idea that DAHP for tryptophan synthesis is channeled via the aro multienzymesystem, we treated conidia with nitrosoguanidine and carried out a filtration enrichment for a trp auxo-troph in a medium containing high concentrations of Phe and Tyr (with the idea that the other two synthaseisozymes would be inhibited). We then scored for trp auxotrophs that could grow also on quinic acid. Theidea was that any trp auxotroph, that could not grow on minimal medium (in addition to no growth on minimalplus high concentrations, 400 µg/ml, of Phe and Tyr) but could grow on quinic acid, must have its lesionprior to dehydroquinic acid and should be a "channeling mutant" with a defective DAHP synthase (trp) isozyme.A long shot idea perhaps, and probably a very naive thing to try, but we were able to find such a mutant.Its properties were the following. It was a non-leaky, it did not grow on Vogel's minimal. It was forall appearances a typical tryptophan auxotroph, responding well to the usual 20 to 40 µg/ml supplement oftryptophan. However it also grew well on Phe and on Tyr, but not at all when both Phe and Tyr were present,and it grew well on a 100 µg/ml supplement of quinic acid. Thus, we had it. A channeling mutant for thearo pathway! We then assayed for DAHP synthase (trp) expecting to find it inactive. To our surprise (andgreat disappointment) it had wild type levels of DAHP synthase (trp). After this, and for reasons which Ino longer remember, we tried to grow the mutant on other amino acids (no response) and also tried niacin.This was all done before I knew anything of nt mutants. To my surprise, niacin permitted growth even betterthan tryptophan. To make a long story short, our so called channeling mutant was none other than one ofthe old nt mutants, now called nt 295 (FGSC #2438 A and #2439 a). It is, perhaps, the only known exampleof a niacin auxotroph isolated by filtration enrichment.My hypothesis, based on the growth data and other evidence, for the rather bizarre behavior of the ntmutant goes like this. Just as there are two kynureninase-type enzymes in N. crassa, one constitutive (ahydroxykynureninase) for the synthesis of NAD and one inducible (a kynureninase) for the degradation oftryptophan (1971 J. Bact. 108: 902), there are two tryptophan oxygenases, a constitutive biosynthetic oneand an inducible catabolic one. The nt mutants, then, lack the constitutive biosynthetic enzyme. Hencethey have a requirement for niacin when grown on minimal medium. They show no such requirement when tryp-tophan is added because tryptophan acts as the inducer for the inducible tryptophan oxygenase which thentakes over for the defective constitutive enzyme. The nt mutants also grow on quinic acid, not becausethey have a lesion prior to dehydroquinic acid, but because quinate by-passes the feedback regulatory con-trols and allows for an accumulation of tryptophan and a subsequent induction of the inducible tryptophanoxygenase. They also grow on Phe or Tyr because Phe or Tyr alone don't adequately block the activities ofthe DAHP synthase isozymes, but do apparently allow for some accumulation of chorismate and hence of tryp-tophan with its subsequent inductive action on tryptophan oxygenase. (Phe and Tyr together, on the otherhand, do adequately block carbon flow via DAHP synthase.)I think this hypothesis can adequately explain what we know about nt mutants. But it is still a hypo-thesis. A major problem in testing it is the lack of assayability of tryptophan oxygenase in the fungi.Why can't this enzyme be assayed in these organisms when it is easily assayed in all others including in-sects, animals and bacteria? The answer in not known. Pete Matchett got so frustrated he suggested thatthe reaction might be nonenzymatic (1974 J. Bact. 118: 837). He showed very clearly the presence ofN-formylkynurenine (the product of the tryptophan oxygenase reaction) in extracts of tryptophan-grownNeurospora, but despite repeated and systematic attempts to assay the enzyme in vitro was unable to detectits activity. Also, Matchett concluded in an earlier report that nt mutants were deficient in tryptophanoxygenase (1970 J. Bact. 103: 364).P.S. In response to Dorothy Newmeyer's question about kynurenine foramidase, nt mutants carry normallevels of this constitutive activity (our unpublished data). Why niacin auxotrophs lacking formamidasehave not been isolated is not known, I can't say. Perhaps there is more than one enzyme or perhapsformamidase has other functions and the mutants are lethal? It is also interesting that the foramidaseof N. crassa is constitutive, whereas it is inducible in bacteria. (This research was sponsored by theOffice of Health and Environmental Research, U.S Department of Energy Contract W-7405-eng-26 with UnionCarbide.) - - - - The University of Tennessee - Oak Ridge Graduate School of Biomedical Sciences andBiology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830.

A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase

F. A. Gaertner

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A response to a letter from David Perkins concerning the question of whether nt mutants involve tryptophan pyrrolase

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