A Common Origin for Enzymes Involved in the Terminal Step

Home , Threonine, Threonine synthase

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, correlates well with some oftheir catalytic properties and indicates RESULTS that they have evolved from a common ancestor. The evolutionary 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, both tryptophan synthase /3 Downloaded by guest on September 24, 2021 Biochemistry: Parsot Proc. Nati. Acad. Sci. USA 84 (1987) 5209

THREONINE SYNTHASE (8.SUBTILIS ) 1MWKGLIHQYKEFLPVTDQTPALTLHEG ,-__, -, , 4 * TRYPTOPHAN SYNTHASE (B.SUBT.ILISP-MYPYPNEIGRYGDFGGKFVPETLMQPLDEIQTAFKOIKDDPAFREEYYKLLKDYSG

N.TPLIHLPKLSEOL.GIELHVKTEGVNPTGSFKDRGMVMAVAKAKEEGNDTIMCAST.GNTSAAAAAYAARANMKC IVIIPNGKIAFGKL 4-4 +++*,,, + +* 4 .4 44* * * _ * 4.- + * ** 4*. --4 *++ +* * RPTALTYADRVTFYLGGAKIYLKREDLNHTGSHKINNALGOALLAKKMGKTKIIAETGAGOHGVAPATVAAKFGFSCTVFMGEEDVAROSL

AOAVMY..GAEIIAIDGNFD...... DALKIVRSICEKSPIALVNSVNPY...... RIEGQKTAAPEVCEOLGE *4++-+ ,+*,, 4 + 4 4 *4*4 _+ * $ NVFRMKLLGAEVVPVTSGNGTLKDATNEAIRYWVOHCEDHFYMI GSVVGPHPYPQVVREFOKMIGEEAKDQLKRIEGTM PDKVVACVGGGS

APDVL A I PV GNAGN ITAYWKGFKEYHEKNGTGLPKMRGF EAE GAAA I VRNE V I ENPE T I ATA. IR IGNP AS WDKAVKAAEE SNGK I .DEV * -.+ __ , ._ * - * * 4 - ++ +, NAMGMF(AFLNEDVELIGAEAAGKGIDTPLHAATISKGTVGVIHGSLTYLIODEFGQIJEPYSISAGLDYPGIGPEHAYLHKSGRVTYDSI

TDDEILHAYQLIARVEGVFAEPGSCASIAGVLKOVKSGEIPKGSKVVAVLTGNGLKDPNTAVDISEIKPVTLPTDEDSILEYVKGAARV *4, +* *+4, _ *+. * ,4--,4 - +.+,, + * 4* * TOEEAVDALKLLSEKEGILPAIESAHALAKAFKLAKGMD..RGQLILVCLSGRGDKDVNTLMNYLEEEVKRHV FIG. 2. Comparison of B. subtilis tryptophan synthase and threonine synthase. The entire amino acid sequences of B. subtills threonine synthase (4) and B. subtilis tryptophan synthase (7) are presented in the one-letter code. They have been aligned by introducing gaps (indicated by dots in the sequences) to maximize the similarity. Asterisks between the sequences indicate identical residues and plus signs indicate conservative replacements: R-K, S-T, D-E, and I-L-V-M-F. Hyphens indicate that the corresponding residue of tryptophan synthase from E. coli, S. cerevisiae, or P. aeruginosa (see ref. 8) is identical to the residue of B. subtilis threonine synthase. Arrow indicates the lysine residue of tryptophan synthase, which binds pyridoxal phosphate (8).

chain and threonine synthase are pyridoxal phosphate-de- synthase 8 chain, and either a y-elimination and a P- pendent enzymes involved in reactions at the p-carbon of replacement or an a- and p-elimination for the threonine amino acid substrates: either a A-elimination or a y-elimina- synthase activity and for the threonine dehydratase activity tion and a B-replacement for the serine dehydratase activity of threonine synthase. These reactions proceed by similar and for the tryptophan synthase activity of tryptophan intermediates: a pyridoxal phosphate Schiff base of a-amino- CH3 HCOH HCNH2 COOH THREONINE THREONINE SYNTHASE A OP03H2 THREONINE I THREONINE SYNTHASE DEHYDRATASE 13 J CH3 CH3 CH2 NC CN2 HCNH2 HN (PLP-ENZ) Ct

COOH COON COON

PHOSPHO- U- KE TOBUT YR ATE HOMOSERINE l m z TR YPTOPHAN a SYNTHASE

NH2 NC HCNH2 C.OOH VINYL LYYCI NE

FIG. 3. Schematic representation of the substrates and products and of one of the reaction intermediates in some of the reactions catalyzed by threonine synthase and tryptophan synthase. The structure ofthe pyridoxal phosphate (PLP) Schiffbase of a-aminocrotonate, an intermediate in each of the reactions catalyzed by threonine synthase (11, 12), threonine dehydratase (13), and tryptophan synthase f8 chain (14) is shown in the box. The pyridoxal phosphate-catalyzed tautomerization of the double bond from ,-y to a-,8 carbons involved in some of these reactions is not presented. Inorganic phosphate, ammonia, and water, the by-products of the reactions, are omitted for clarity. Downloaded by guest on September 24, 2021 5210 Biochemistry: Parsot Proc. Natl. Acad. Sci. USA 84 (1987) acrylate (for tryptophan synthase) or a-aminocrotonate (for perform the primary reactions catalyzed by the present-day threonine synthase). Moreover, tryptophan synthase can threonine synthase and tryptophan synthase remains specu- catalyze the conversion of 2-amino-3-butenoic acid (vinyl- lative. It is also probable that some ofthe reactions catalyzed glycine; see Fig. 3) to a-ketobutyrate and ammonia via by the ancestral enzyme could have yielded deleterious formation of an a-aminocrotonate Schiff base intermediate metabolites such as the product of the condensation of indole (14). Tryptophan synthase P2 can thus lead to the same with a-aminocrotonate. Gene duplication followed by muta- reaction intermediate as that found in the threonine synthase tions leading to specialization in the substrate specificity of and threonine dehydratase reactions and can yield the same the encoded proteins may thus have been selected not only product (Fig. 3). In addition, threonine synthase catalyzes a to improve the efficiency of the enzyme in a given way, but reaction similar to and shares amino acid sequence homology also to eliminate unwanted reactions. hi the case of with threonine dehydratase (4), which, like tryptophan tryptophan synthase , chain, such a specialization could also synthase, is also endowed with serine dehydratase activity. have involved its close association with the a chain, chan- Interestingly the lysine residue of tryptophan synthase, neling indole into the catalytic site of the p subunit and which binds pyridoxal phosphate (8), is present in a region preventing its serine dehydratase activity (9, 10). conserved not only in threonine synthase (arrow in Fig. 2) but also in threonine dehydratase and in D-serine dehydratase (4). I am grateful to M. Goldberg, P. Marliere, G. N. Cohen, B. No common feature was detected between the sequences of Davidson, and A. Pugsley for a critical reading ofthe manuscript, and the pyridoxal phosphate binding sites of various other en- to B. Caudron for advice in the use ofcomputer tools. This work was zymes (15). Therefore, the conservation of this region in the supported by the Centre National de la Recherche Scientifique threonine synthase and tryptophan synthase p-chain se- (Unite Associee 1129). quences strongly supports the relationship between these 1. Horowitz, N. H. (1945) Proc. Natl. Acad. Sci. USA 31, enzymes. 153-157. The similarity in the catalytic mechanisms and the high 2. Jensen, R. A. (1976) Annu. Rev. Microbiol. 30, 409-425. similarity in primary structures indicate a common evolu- 3. Belfaiza, J., Parsot, C., Martel, A., Bouthier de la Tour, C., tionary origin for threonine synthase and tryptophan syn- Margarita, D., Cohen, G. N. & Saint-Girons, I. (1986) Proc. thase. This is in contrast to other enzymes, such as trypto- Nati. Acad. Sci. USA 83, 867-871. phanase and tryptophan synthase, which, while catalyzing 4. Parsot, C. (1986) EMBO J. 5, 3013-3019. similar reactions, do not share any sequence similarity (6, 5. Lipman, D. J. & Pearson, W. R. (1985) Science 227, 1435-1441. 16). 6. Crawford, I. P., Nichols, B. P. & Yanofsky, C. (1980) J. Mol. The evolutionary relationship demonstrated here between Biol. 142, 489-502. enzymes involved in the terminal step of two biosynthetic 7. Henner, D. J., Band, L. & Shimotsu, H. (1984) Gene 34, pathways as different as those of tryptophan and threonine 169-177. was unexpected. This observation strongly supports Jensen's 8. Hadero, A. & Crawford, I. P. (1986) Mol. Biol. Evol. 3, hypothesis (2) that ancestral organisms produced a relatively 191-204. small number of enzymes with flexible substrate specificity 9. Yanofsky, C. & Crawford, I. P. (1972) in The Enzymes, ed. acting in several metabolic pathways. In the case of the Boyer, P. D. (Academic, New York), Vol. 7, 3rd Ed., pp. threonine synthase, threonine dehydratase, D-serine dehy- 1-31. dratase, and tryptophan synthase p-chain family, the ances- 10. Miles, E. W. (1979) Adv. Enzymol. Relat. Areas Mol. Biol. 49, tral enzyme may have been endowed with many activities 127-186. 11. Flavin, M. & Slaughter, C. (1960) J. Biol. Chem. 235, centered around the pyridoxal phosphate-dependent stabili- 1112-1118. zation of a-aminoacrylate or a-aminocrotonate intermedi- 12. Skarstedt, M. T. & Greer, S. B. (1973) J. Biol. Chem. 248, ates. Although threonine synthase, threonine dehydratase, 1032-1044. and tryptophan synthase p chain primarily catalyze different 13. Phillips, A. T. & Wood, W. (1965) J. Biol. Chem. 240, classes of reactions, they are still endowed with common 4703-4709. overlapping activities such as the threonine dehydratase 14. Miles, E. W. (1975) Biochem. Biophys. Res. Commun. 66, activity of threonine synthase, the serine dehydratase activ- 94-102. ity of threonine dehydratase and tryptophan synthase p 15. Tanase, S., Kojima, H. & Morino, Y. (1979) Biochemistry 18, chain, and the p-elimination reaction catalyzed by trypto- 3002-3007. phan synthase p chain on the 4-carbon substrate vinylglycine. 16. Deeley, M. C. & Yanofsky, C. (1981) J. Bacteriol. 147, Whether or not the ancestral enzyme was readily able to 787-796. Downloaded by guest on September 24, 2021