Tryptophan biosynthesis: The material that follows is not included in the main part of chapter 6. However, much of the pathway that is involved is illustrated in figure 6.15, which goes with problem 13. The tables that follow were employed by Susan Dutcher in the version of MCDB 2150 that she taught at the time I first began teaching this course. Auxotrophic mutations that require tryptophan for growth were tested for their ability to grow on a series of known precursors for the biosynthesis of tryptophan. Mutants were found that were blocked at five different steps leading from chorismate (the first precursor in the series) to tryptophan. The patterns of which intermediates would or would not support growth of each mutant strain agreed exactly with the known pathway for biosynthesis of tryptophan. The overall pathway of tryptophan biosynthesis in Neurospora is chorismate --> anthranilate --> phosphoribosyl anthranilate --> 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate --> indoleglycerol phosphate --> tryptophan. The enzymes involved in each step are shown below in italics. A crudely simulated arrow head (\:/) has been used to depict the reaction arrow for each catalytic step because of lack of an appropriate vertical arrow symbol in html.
Growth characteristics of mutants: The table that follows
shows the growth of seven mutant strains (M1 - M7) of Neurospora
on TRP and the its biosynthetic intermediates.
Complementation analysis was done by fusing hyphae such that the cultures
contained nuclei with two different types of mutations. In cases where
each nucleus was able to code for the enzyme whose gene was mutated in
the other nucleus, phenotypically wild type growth was obtained. Analysis
of the seven mutant strains yielded the results
shown in the table below (mut = mutant phenotype, requiring tryptophan
for growth; wt = wild-type phenotype, capable of growing without
added tryptophan or any of its biosynthetic intermediates):
Conclusions: All seven mutations fail to complement themselves, as expected. Mutations 3 and 5 fail to complement each other, indicating that both affect the same peptide chain in the enzyme, anthranilate phosphoribosyl transferase, which is needed for synthesis of P-ANTH. Mutations 4 and 7 do complement each other, despite the fact that both cause loss of tryptophan synthase activity. This is consistent with the fact that tryptophan synthase is known to be composed of two subunits designated alpha and beta. Thus, complementation analysis precisely verifies the biosynthetic pathway and allows identification of mutations that affect the same step as well as identification of enzymes that appear to be composed of two or more subunits.
Suicide substrates: Genetic studies on tryptophan biosynthetic pathways have also been done in Arabadopsis, a small flowering plant. In this case, a suicide substrate was used to select for mutant strains. The enzymes that convert anthranilate to tryptophan can also convert 5-methylanthranilate to 5-methyltryptophan, which is incoprorated into proteins like tryptophan, but results in non-functional proteins, such that the plant does not survive. 5-methyltryptophan also blocks de novo synthesis of tryptophan by feedback inhibition of anthranilate synthase, thus preventing biosynthetic tryptophan from competing with the 5-methyltryptophan. Plants with mutations blocking any steps in the pathway between anthranilate and tryptophan can thus grow in the presence of 5-methylanthranilate when also supplied with small amounts of tryptophan, whereas those that are not blocked will make enough 5-methyltryptophan to inhibit growth even in the presence of the small amount of added tryptophan. This provides a powerful way to select for tryptophan auxotrophs in Arabadopsis.