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A Common Origin for Enzymes Involved in the Terminal Step

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A Common Origin for Enzymes Involved in the Terminal Step Proc. Natl. Acad. Sci. USA Vol. 84, pp. 5207-5210, August 1987 Biochemistry A common origin for enzymes involved in the terminal step of the threonine and tryptophan biosynthetic pathways (threonine synthase/tryptophan synthase/metabolic pathway/evolution/pyridoxal phosphate) CLAUDE PARSOT Unite de Biochimie Cellulaire, Ddpartement de Biochimie et Gdndtique Moldculaire, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris, Cedex 15, France Communicated by Edmond H. Fischer, April 24, 1987 (received for review February 19, 1987) ABSTRACT Comparison of the amino acid sequence of sequence present in a given library. The statistical signifi- Bacillus subtilis threonine synthase with the National Biomed- cance for the comparison of a pair of proteins was tested ical Research Foundation protein sequence library revealed a using the RDF computer program (5). statistically significant extent of similarity between the se- The protein library of the National Biomedical Research quence of the tryptophan synthase f chain from various Foundation was used as the amino acid sequence data base. organisms and that ofthreonine synthase. This homology in the To avoid insignificant signals, the protein library was restrict- primary structure of threonine synthase and tryptophan ed to the sequence of enzymes (618 sequences). The results synthase j8 chain, which catalyze the last step in the threonine were then checked using the whole data base. and the tryptophan biosynthetic pathways, respectively, cor- relates well with some oftheir catalytic properties and indicates RESULTS that they have evolved from a common ancestor. The evolu- tionary relationship between these enzymes supports the hy- The computer program FASTP (5) was used to compare the pothesis that primitive enzymes possessed a broad substrate sequence of Bacillus subtilis threonine synthase (4) with the specificity and were active in several metabolic pathways. sequences of all the enzymes in the National Biomedical Research Foundation protein library. The histogram of the One of the difficulties in understanding the mechanisms initial scores for the 618 comparisons is shown in Fig. 1. B. whereby multistep metabolic pathways have evolved is the subtilis threonine synthase is clearly more closely related to apparent lack of any selective advantage conferred by indi- the tryptophan synthase ,3 chains from Salmonella typhimu- vidual steps of the pathway prior to the establishment of the rium and Escherichia coli (with similarity values of64 and 55, whole pathway. Horowitz (1) proposed that evolution of such respectively) than to all other enzymes in the library, includ- pathways proceeded in a stepwise manner, with individual ing E. coli threonine synthase (similarity value of 39). The steps being recruited in the reverse direction relative to the other sequence showing similarity with B. subtilis threonine final pathway-i.e., the last step in a pathway was acquired synthase is that of Saccharomyces cerevisiae threonine first, the penultimate step next, and so on. From consider- dehydratase, as previously noted (4). On the other hand, the ations of the substrate ambiguity exhibited by contemporary sequence of S. cerevisiae tryptophan synthase / chain, enzymes, Jensen (2) suggested an alternative hypothesis that although present in the library, is not clearly resolved from primitive enzymes possessed a very broad specificity, per- the sequences of the bulk of the other enzymes. The se- mitting them to utilize a wide range of structurally related quences of B. subtilis and Pseudomonas aeruginosa trypto- substrates, thereby yielding small amounts of erroneous phan synthase B chain were not present in the library. After products. This process would have provided a biochemically optimization (5), the similarity values for the comparison of diverse environment in which individual enzyme recruitment B. subtilis threonine synthase with S. typhimurium, E. coli would improve the function of a slow, but already existing, tryptophan synthase /3 chain and E. coli threonine synthase multistep sequence. amino acid sequences were 86, 77, and 100, respectively, Jensen's proposal is supported by the recent discovery of confirming that the closest relationship is between the thre- sequence similarity between enzymes catalyzing consecutive onine synthases, as might be expected. steps in the methionine pathway (3) and also To ascertain the statistical significance of the similarity biosynthetic detected between B. subtilis threonine synthase and S. between two enzymes catalyzing consecutive steps in the typhimurium tryptophan synthase p chain, the two sequences isoleucine biosynthetic pathway (4). Here, I report amino were compared using the RDF computer program (5). Com- acid sequence comparisons that suggest a common evolu- parison ofB. subtilis threonine synthase with S. typhimurium tionary origin for threonine synthase and tryptophan syn- tryptophan synthase p chain (6) and with 100 randomly thase /8 chain, the enzymes involved in the last step of the permuted versions of the latter gave, for the comparison of threonine and tryptophan biosynthetic pathways, respective- the two initial sequences, a similarity value corresponding to ly. The sequence similarity detected correlates well with 7.8 SD above the mean of random sequences. According to similarities in some catalytic properties of these enzymes, Lipman and Pearson (5), this value indicates a significant despite the fact that the pathways in which they act are degree of relatedness. unrelated. Fig. 2 shows the alighment of the B. subtilis threonine synthase and B. subtilis tryptophan synthase /3 chain (7) MATERIALS AND METHODS sequences: 9 gaps have been introduced in the sequences, 2 of which account for a total of 29 positions, the 7 others for The computer program FASTP of Lipman and Pearson (5) 11 residues. Of the remaining 334 compared positions, 71 are was used to compare individual sequences with each protein occupied by identical residues (21%) and 48 by conservative replacements (as defined in legend of Fig. 2), which gives a The publication costs of this article were defrayed in part by page charge score of 36% similarity. In addition, there are 20 positions payment. This article must therefore be hereby marked "advertisement" where an amino acid residue of B. subtilis threonine in accordance with 18 U.S.C. §1734 solely to indicate this fact. synthase, being neither identical nor homologous to the 5207 Downloaded by guest on September 24, 2021 5208 Biochemistry: Parsot Proc. Natl. Acad. Sci. USA 84 (1987) number of sequences .thr. synthase (E. coil) thr. dehydratase (S. cerevisiae) trp. synthase (E. coli ) rWtrp. synthase similarity 10 value FIG. 1. Histogram of the similarity values for the comparison of the B. subtilis threonine synthase with 618 enzyme sequences. Comparison was performed with the FASTP computer program of Lipman and Pearson (5) against enzyme sequences in the National Biomedical Research Foundation protein library. The similarity value for each comparison is given on the x axis, and the number of sequences detected at each similarity value is indicated on the y axis. Arrows indicate the proteins with a high score of similarity with B. subtilis threonine synthase. The sequence detected at a similarity value of 42 was that of RNA-directed RNA polymerase chain from bacteriophage MS2, but the similarity encompassed only 23 residues and was not significant. relevant residue in B. subtilis tryptophan synthase chain, is second, the condensation of indole onto L-serine to yield identical to an amino acid residue present at the same position L-tryptophan, which can be accomplished by a P2 dimer. In in either E. coli, S. typhimurium, P. aeruginosa, or S. addition, the /32 dimer catalyzes the deamination of L-serine, cerevisiae tryptophan synthases, as aligned by Hadero and producing pyruvate and ammonia. In this reaction, a Schiff Crawford (O. Thus, -42% of the residues of the B. subtilis base intermediate of a-aminoacrylate is formed between threonine synthase and tryptophan synthase /3 chain are enzyme-bound pyridoxal phosphate and L-serine. This inter- identical or closely related after deletions and insertions have mediate can yield either pyruvate and ammonia, via a been excluded. /3-elimination reaction, or L-tryptophan via a y-elimination and a /3-replacement reaction with indole. DISCUSSION Threonine synthase catalyzes the conversion of phospho- homoserine to threonine and inorganic phosphate. The re- The similarity detected between the primary structures of action proceeds by the formation of a pyridoxal phosphate threonine synthase and tryptophan synthase may reflect (in Schiffbase intermediate of a-aminocrotonate, to which water part) the similar catalytic mechanisms of these enzymes. is added to form threonine (11). In addition, threonine Properties oftryptophan synthase have been comprehensive- synthase is endowed with a low threonine dehydratase ly reviewed by Yanofsky and Crawford (9) and by Miles (10). activity (12); this latter reaction, like that catalyzed by This enzyme is an a2/32 heterotetramer, which catalyzes the threonine dehydratase (13), is thought to involve the same following reactions: first, the hydrolysis of indole-3-glycerol a-aminocrotonate intermediate, which is converted to a- phosphate to give indole and D-glyceraldehyde 3-phosphate, iminobutyrate and spontaneously hydrolyzes to a-ketobuty- a reaction that can be performed by the a subunit alone; and rate and ammonia (Fig. 3). Thus,
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