*Department of Chemistry, Duquesne University, Pittsburgh, Pennsylvania 15219; tDepartment of Biological Chemistry, California College of Medicine,.
Proc. Natl. Acad. Sci. USA Vol. 76, No. 4, pp. 1589-1592, April 1979 Biochemistry
Amino acid sequence of ATP phosphoribosyltransferase of Salmonella typhimurium (histidine operon/autogenous regulation/sequence homologies)
DENNIS PISZKIEWICZ*, BILL E. TILLEYt, TSAFRIRA RAND-MEIRt, AND STANLEY M. PARSONSt *Department of Chemistry, Duquesne University, Pittsburgh, Pennsylvania 15219; tDepartment of Biological Chemistry, California College of Medicine, University of California, Irvine, California 92717; and tDepartment of Chemistry, University of California, Santa Barbara, California 93106
Communicated by Thomas C. Bruice, January 8, 1979
ABSTRACT The amino acid sequence of ATP phosphoribosyltransferase [1(5'-phosphoribosyl)ATP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.17] of Salmonella typhimurium has been determined. The amino acid sequence analysis was carried out with a combination of manual and automated methods. It was complemented by DNA sequence analysis (done in another laboratory) of the hisG gene, which codes for it. The subunit polypeptide chain contains 299 amino acid residues and has a molecular weight of 33,216. The amino-terminal segment of the protein is relatively basic in character and has limited sequence homologies with the lac repressor and histidinol dehydrogenase. In addition, the protein contains a 40-residue segment that has 13 residues identical with the sequence surrounding the active-site cysteine of glyceraldehyde-3-phosphate dehydrogenase. The first enzyme of the histidine biosynthetic pathway in Salmonella typhimurium is ATP phosphoribosyltransferase
[1-(5'-phosphoribosyl)-ATP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.17] (1, 2). It catalyzes the transfer of the phosphoribosyl moiety from phosphoribosyl pyrophosphate to ATP to form phosphoribosyl-ATP and inorganic pyrophosphate (1, 2). This enzyme plays a central role in the regulation of histidine metabolism. It has been shown to interact with every molecular regulatory signal known to affect expression of the histidine operon (3). This enzyme regulates histidine biosynthesis at the level of catalysis by being feedback inhibited by the end product, histidine (4). Furthermore, it is an autogenous (5) or autoregulatory (6) protein by virtue of regulating the expression of its own gene. Missense mutants of the enzyme that affect sensitivity to inhibition by histidine also alter expression of the histidine operon in vivo (2). The purified enzyme binds DNA of the regulatory region of the histidine operon (7) and inhibits transcription of the operon in vitro (8, 9). The DNA sequences of the histidine operon control regions of Salmonella typhimurium (10) and Escherichia coli (11) are now known. ATP phosphoribosyltransferase has a molecular weight of approximately 215,000 (12). It is composed of six identical polypeptide chains having molecular weights of between 33,000 (13) and 35,000 (12). The subunit polypeptide chain is coded for by the hisG gene, the first gene of the histidine operon of Salmonella typhimurium (1, 2). As a first step in defining structure-function relationships of this protein, we have determined the amino acid sequence of its subunit polypeptide chain. We now report the sequence of the 299 amino acids of the subunit of ATP phosphoribosyltransferase.
MATERIALS AND METHODS ATP phosphoribosyltransferase of Salmonella typhimurium was prepared by the method of Parsons and Koshland (13). Before sequence analysis it was denatured in 6 M guanidine hydrochloride, reduced, and carboxymethylated (14). Iodo[3H]acetic acid, from New England Nuclear, was used as the alkylating agent to facilitate identification of cysteine residues. The amino-terminal sequence of the protein was determined by automated Edman degradation on a Beckman model 890B sequencer. The methods used and the results obtained have
been published (15). Peptides were derived from the protein by cleavage with trypsin from Worthington (16), staphylococcal protease from Miles (17), and cyanogen bromide. They were initially segregated according to size by gel filtration on Sephadex G-50, then fractionated further by column chromatography on Dowex 1, paper chromatography, and high-voltage paper electrophoresis. Sequences of the larger peptides were determined by automated Edman degradation with a Beckman sequencer; phenylthiohydantoin derivatives were identified by gas chromatography and thin-layer chromatography (18). All manual sequencing methods used were those described earlier (16). Details of the peptide isolation and sequence determination will be published elsewhere. RESULTS AND DISCUSSION The amino acid sequence of ATP phosphoribosyltransferase is given in Fig. 1. The amino-terminal sequence of the protein was determined by automated Edman degradation (15). Peptides were derived from the protein by cleavage with trypsin, staphylococcal protease, and cyanogen bromide. The complete amino acid sequence has been accounted for with polypeptide fragments, and these were overlapped to reconstruct the complete sequence (Fig. 1). During the course of this study, DNA sequence data on the hisG gene (unpublished data) were made available to us by Wayne Barnes and R. N. Husson. This information greatly speeded our project by allowing us to order many polypeptide fragments derived from the protein. Most of these were subsequently verified by amino acid sequencing data. At the present time the amino acid sequence relies on DNA sequencing data to overlap positions 79-80, 80-81, 184-185, and 186-187. The subunit polypeptide chain of ATP phosphoribosyltransferase contains 299 amino acids. Its molecular weight in the un-ionized form calculated from the sequence is 33,216; this compares favorably with the values of 33,000 (13) to 35,000 (12)
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1589
Biochemistry: Piszkiewicz et al.
1590 I
Proc. Natl. Acad. Sci. USA 76 (1979)
20 10 "- ^_4-u-ThrA~q-aLAfl-j4A4-j-Gl n-LT;Ser- I r-Arq- -GI n-Asp- 4Ser-Arg-GI
30
u-Leu-L%-Al a-Arg-C4-Gly;I I e-
40 50 60 Lys- II e-Asn-Leu-Hi s-Thr-Gl n-Arg-Leu- II e-Ala-Met-Al a-Gl u-Asn-Met-Pro- Il e-Asp- I 1e-Leu-Arg-Val -Arg-Asp-Asp-Asp- I 1e-Pro-Gly-
T9~~~~~~~N~3.
TB
CNBr4
SP2
SP3
70 80 90 Leu-Va 1 -Met-Asp-Gly-Vai -Vii -Asp-Leu-Gly- I1e-Il e-Gly-Gl u-Asn-Val -Leu-Gl u-Gl u-Gl u-Leu-Leu-Asn-Arg-Arg-Ala-Gl n-Gly-Gl u-AspT13
SP
SP3
SP5+6
>
>*
T12+13
SP8+9
110
100
120
Pro-Arg-Tyr-Leu-Thr-Leu-Arg-Arq-Leu-Asp-Phe-Gly-Gly-Cys-Arg-Leu-Ser-Leu-Al a-Thr-Pro-Val -Asp-GJ u-Al a-Trp-Asp-Gly-Pro-Al a> < > o < T14 >< T16 T17+18 C11BrS
SP8+9
SP9
130
_
140
SPI 0
-t
CNBrs
121 ..X
T22
spil 170
160
180
Leu-Asn-Gly-Ser-Val -Gi u-Val -Ala-Pro-Arg-Ala-Gly-Leu-Al a-Asp-Ala - Il e-Cys-Asp-Leu-Val -Ser-Thr-Gly-Al a-Thr-Leu-Gl u-Al a-AsnT24
T23
CNBr5 S < T30 > > . > U32L > 133 CNBr6
___
SPi7
>
__ __SP__a 260
250
270
Va 1- I e-AI a-Leu-Leu-Pro-GI y-'Aia-GI u-Arg-Pro-Thr- I Ie-Leu-Pro-Leu-Al a-GI y-Gl u-Gl n-Gl n-Arg-Val -Ala-Met-HI s-Met-VaI -Ser-Ser-
T34
CNBr8
S>
SP19
eCIIrt3
T r35 T37 38 < CNBrl 0 CNBrl I SP21
FIG. 1. Amino acid sequence of ATP phosphoribosyltransferase from Salmonella typhimurium. Double-headed arrows under the sequence represent peptides derived from the protein and used to reconstruct its sequence; under amino acids indicates residues identified by automated Edman degradation. The identities of residues 20, 25, and 28 were determined by this method although not reported earlier (15). Cleavage methods used are: T, trypsin; SP, staphylococcal protease; CNBr, cyanogen bromide. Peptides CNBr2 and CNBr5 have had only their compositions and amino-terminal residues determined to date.
obtained by physical methods. The amino-terminal residue is methionine, and comparison of the amino terminus with the structure of the hisG gene (10) indicates that only the N-formyl group has been removed after protein synthesis. The enzyme has five cysteines per subunit (Fig. 1), the same number as can be titrated in the native enzyme by bis-(5-carboxyl-4-nitro-
phenyl)disulfide (19). Therefore, in the native enzyme all cysteines are in the reduced form; there are no disulfide bridges. There are no obvious sequences within the protein that are repeated. There are no large clusterings of hydrophobic or hydrophilic amino acid residues. However, there does appear to be a clustering of basic amino acid residues near the amino
1591
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Piszkiewiez et al. ATP Phosphoribosyltransferase
Met-Leu-Asp-Asn-Thr-Arg-Leu-Arg- lie-Ala-Il e-Gl n-Lys-Ser-Gly-Arg-Leu-Gln-Asp-Asp-Ser-Arg-Gl u-
Lac Repressor
Met
Il
Lys-Pro-ValiThre-y-s-a
10 l
luTrAaGyVlSrTr l
20
h-a!e-rVl
50 40 30 24 Leu-Leu-Al a-Arg-Cys-Gly- Il1e-Lys -Il1e-Asn-Leu-Hi s-Thr-Gl n-Arg-Leu- Il1e Al a-Met-Al a-Gl u- sn-Met-Pro- Il1e-Asp- Il1e-Leu50 40 30 24 Val -Asn-Gl n-Ala-Ser-Hi s-Val -Ser-Al a-Lys-Thr-Arg-Gl u-Lys-Val -Gi u-Al a-Al a-Met-Al a-Gl u Leu-Asn-Tyr- Il e-Pro-Asn-Arg-
mi
FIG. 2. Comparison of the amino-terminal sequences of ATP phosphoribosyltransferase and the lac repressor (27).
terminus of the protein, followed by a clustering of acidic groups. The net charge on residues 1-40 is +6, and on residues 41-80 it is -9. The preponderance of basic groups near the amino terminus suggests that this region might be involved in binding nucleic acids, as is the case with the histones (20). When the aminoterminal 50 residues of ATP phosphoribosyltransferase are aligned with histone 2a (21), histone 2b (22), histone 3 (23), or histone 4 (24), identical residues are found at three, one, one, and two positions, respectively. It is rare to find as much as 10% identity in unrelated sequences of length greater than 25 residues (25), and homologies are considered significant when at least 20% of the amino acids in two aligned sequences are identical.§ Consequently, it can be concluded that the transferase and histones are unrelated. The amino-terminal sequence of ATP phosphoribosyltransferase was also compared with the § Baker, W. C. & Dayhoff, M. 0. (1970) in Biophysical Society Abstracts, 14th Annual Meeting, p. 152a.
HH
II
amino-terminal sequences of numerous other proteins that interact with nucleic acids (26). Only one of these proteins showed identities numerous enough to be considered significant. The amino-terminal sequence of ATP phosphoribosyltransferase is identical at 11 of 48 residues, 22% of the amino terminus of the lac repressor (27) (Fig. 2); sequence homologies beyond residue 48 appear to be no greater than would be expected on the basis of chance. The 51-residue segment at the amino terminus of the lac repressor has been implicated in its interaction with lac operator DNA (28). On the basis of the basic character of this region and the admittedly limited sequence homology with the lac repressor, one might speculate that the amino terminus of ATP phosphoribosyltransferase interacts with histidine operator DNA while mediating expression of the histidine operon. In an earlier report (15) we noted a sequence homology of the amino-terminal segments of ATP phosphoribosyltransferase and histidinol dehydrogenase (29), the second gene product of the histidine operon and the terminal enzyme of the histidine
Il~~~~~40IH III ~
i5oH 1
14*1150
II
ATP Phosohoribosyltransferase
-Leu-Leu-Lys-Arg-
-Tyr-Leu-Asp-Gl n-Lys-Gly-Val-Ser-Phe-Lys-Ser-Cys-Leu-Leu-Asn
Pig GPDH
-His-Gl u-Lys-Tyr-
-Asp-Asn-Ser-Leu-Lys-Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-
Lobster GPDH
-Leu-Gl u-Lys Tyr-
-Ser-Lys-Asp-Met-Thr-Val -Val -Ser-Asn-Al a Ser-Cys-Thr-Thr -Asn-
Yeast GPDH
(Ser,Glu,Lys)Tyr-
-Thr-Ser-AsplLeuJLys-Ile-Val -Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-
B. stearothermophilus GPDH
a-Ser-Cys-Thr-Thr-Asn-Glu-Asp-Lys-Tyr-Asp-Pro-Lys-Ala-His-His-Val-Ile-Ser-Asn-Al L 1 Li Li~~~~~~~~~~~~~~~~~~~~~ Li -Gly-Ser-Val -G u-Val -Al Pro-Arg-Al a-Gly-Leu-Al Asp-Al a-Il e-Cys-Asp-Leu-Val -Ser-ThrL.....~ Li170 160
-Cys-Leu-Ala-Pro-Leu-Ala-Lys-Val-Ile-His-Asp-H1s-Phe-Gly-Ile-Val-Glu-Gly-Leu-Met-Thr-Cys-Leu-Al a-Pro-Val -Al a-Lys-Val -Leu-Hi s-G1 u-Asn-Phe-Gl u-Il e-Val -Gl u-Gly-Leu-Met-Thr-Cys-Leu-Al a-Pro-Leu-Al a-Lys-Val -Il e-Asn-Asp-
-Phe-Gly-Il1e-Gl u-Gl u-Gly-Leu-Met-Thr-
-Cys-Leu-Al a-Pro-Phe-Al a-Lys-Val -Leu-Hi s-Gl n-Gl u-Phe-Gly-Il e-Val -Arg-Gly-Met-Met-ThrFIG. 3. Comparison of segments of ATP phosphoribosyltransferase and glyceraldehyde-3-phosphate dehydrogenases (GPDH) from pig (33), lobster (34), yeast (35), and Bacillus stearothermophilus (36). Cysteine-149 residues of the pig, lobster, and yeast dehydrogenases have been shown to be at their active sites (33-35).
1592
Biochemistry: Piszkiewicz et al.
biosynthetic pathway (1, 2). The full extent of .this homology will not be known until the complete amino acid sequence of histidinol dehydrogenase is determined. Sequence similarities between these two enzymes, if of substantial length, could reflect a common ancestral gene in the evolution of these proteins encoded by the histidine-operon. Indeed, in his proposal of the hypothesis of retrograde evolution, Horowitz (30) speculated that, for the histidine operon of Salmonella typhimurium, the gene defining the enzyme catalyzing the last step of the pathway (histidinol dehydrogenase) gave rise to the gene defining the first enzyme (ATP phosphoribosyltransferase). In view of the sequence homologies between ATP phosphoribosyltransferase and the lac repressor and histodinol dehydrogenase, one might ask if there is any relationship between the amino-terminal sequences of the lac repressor and histidinol dehydrogenase. No striking similarities of these two segments can be found. ATP phosphoribosyltransferase is believed to have a cysteine that is essential for activity (31, 32); however, the essential cysteine has not yet been identified. We find that a 40-residue segment of ATP phosphoribosyltransferase that contains a cysteine is homologous with the sequence surrounding the active site cysteine of glyceraldehyde-3-phosphate dehydrogenases from various sources (33-36) (Fig. 3). When cysteine-149 of the transferase is aligned with cysteine-149 of the dehydrogenases (the identical residue numbers are probably fortuitous) 14 of 40 residues (35%) are identical. No other homologies of comparable magnitude were found when the ATP phosphoribosyltransferase sequence was compared with the sequence (26) around active-site cysteine residues of other enzymes. The similarity of amino acid sequences suggests the possibility that their tertiary structures are similar, at least in the homologous regions. Glyceraldehyde-3-phosphate dehydrogenase has as part of its tertiary structure an architectural feature found in many enzymes that catalyze reactions involving ATP or NAD, the NAD-binding domain (also called the dinucleotide fold) (37). Cysteine-149 of the dehydrogenase is the carboxyl-terminal residue of the NAD-binding domain (37). On the basis of the amino acid sequence homology (Fig. 3) we may now ask if cysteine-149 is the residue essential for the activity of ATPphosphoribosyltransferase and if this enzyme has in its tertiary structure the NAD-binding domain, where it might bind ATP or other nucleotides involved in its activities. Note Added in Proof. Residue 18 was originally misidentified as Gln; further studies have shown that it is Ser. This result is in agreement with the DNA sequence of the hisG gene. We thank Dr. Wayne M. Barnes and R. N. Husson for communicating to us during this project their results on the DNA sequence of the hisG gene. This work was supported by Special Grant 799 from the American Cancer Society, California Division, and Grant PCM76-81289 from the National Science Foundation. 1. Brenner, M. & Ames, B. N. (1971) in Metabolic Pathways, eds. Greenberg, D. M. & Vogel, H. J. (Academic, New York), Vol. 5, pp. 349-387. 2. Goldberger, R. F. & Kovach, J. S. (1972) Curr. Top. Cell. Regul.
5,285-308. 3. Kleeman, J. E. & Parsons, S. M. (1977) Proc. Natl. Acad. Sci. USA 74, 1535-1537. 4. Martin, R. G. (1963) J. Biol. Chem. 238,257-268.
Proc. Natl. Acad. Sci. USA 76 (1979) 5. Goldberger, R. F. (1974) Science 183,810-816. 6. Calhoun, D. H. & Hatfield, G. W. (1975) Annu. Rev. Microbiol. 29,275-299. 7. Meyers, M., Blasi, F., Bruni, C. B., Deeley, R. G., Kovack, J. S., Levinthal, M., Mullinix, K. P., Vogel, T. & Goldberger, R. F. (1975) Nucleic Acids Res. 2,2021-2036. 8. Blasi, F., Bruni, C. B., Avitabile, A., Deeley, R. G., Goldberger, R. F. & Meyers, M. M. (1973) Proc. Natl. Acad. Sci. USA 70, 2692-2696. 9. DiNocera, P. P., Avitabile, A. & Blasi, F. (1975) J. Biol. Chem. 250,8376-8381. 10. Barnes, W. M. (1978) Proc. Natl. Acad. Sci. USA 75, 42814285. 11. DiNocera, P. P., Blasi, F., DiLauro, R., Frunzio, R. & Bruni, C. B. (1978) Proc. Natl. Acad. Sci. USA 75,4276-4280.
12. Voll, M. J., Appella, E. & Martin, R. G. (1967) J. Biol. Chem. 242,
1760-1767. 13. Parsons, S. M. & Koshland, D. E., Jr. (1974) ]. Biol. Chem. 249, 4104-4109. 14. Piszkiewicz, D., Landon, M. & Smith, E. L. (1971) ]. Biol. Chem. 246, 1324-1329. 15. Piszkiewicz, D., Rand-Meir, T., Theodor, I. & Parsons, S. M. (1977) Biochem. Biophys. Res. Commun. 78, 833-838. 16. Landon, M., Piszkiewicz, D. & Smith, E. L. (1971) J. Biol. Chem. 246,2374-2399. 17. Drapeau, G. R. (1977) Methods Enzymol. 47, 189-191. 18. Summers, M. R., Smythers, G. W. & Oroszlan, S. (1973) Anal. Biochem. 53, 624-628. 19. Parsons, S. M. & Lipsky, M. (1975) J. Bacteriol. 121, 485-490. 20. Elgin, S. C. R. & Weintraub, H. (1975) Annu. Rev. Biochem. 44, 725-774. 21. Yoeman, L. C., Olson, M. 0. J., Sugano, N., Jordan, J. J., Taylor, C. W., Starbuck, W. C. & Busch, H. (1972) J. Biol. Chem. 247, 6018-6023. 22. Hnilica, L. S., Kappler, H. A. & Jordan, J. J. (1970) Experimentia 26,353-355. 23. De Lange, R. J., Hooper, J. A. & Smith, E. L. (1972) Proc. Natl. Acad. Sci. USA 69,882-884. 24. De Lange, R. J., Fambrough, D. M., Smith, E. L. & Bonner, J. (1969) J. Biol. Chem. 244, 319-334. 25. Hood, M. L., Fowler, A. V. & Zabin, I. (1978) Proc. Natl. Acad. Sci. USA 75, 113-116. 26. Dayhoff, M. 0. (1972) Atlas of Protein Sequence and Structure
(National Biomedical Research Foundation, Washington, DC), Vol. 5 and Suppl. 1 (1973) and 2 (1976). 27. Beyreuther, K., Adler, K., Fanning, E., Murray, C., Klemm, A. & Geisler, N. (1973) Proc. Natl. Acad. Sci. USA 70, 35763580. 28. Jovin, T. M., Geisler, N. & Weber, K. (1977) Nature (London) 269,668-672. 29. Yourno, J. (1971) J. Mol. Biol. 62,223-231. 30. Horowitz, N. H. (1965) in Evolving Genes and Proteins, eds. Bryson, V. & Vogel, H. J. (Academic, New York), pp. 15-23. 31. Bell, R. M. & Koshland, D. E., Jr. (1971) Bioorg. Chem. 1, 409-423. 32. Parsons, S. M. & Lipsky, M. (1975) J. Biol. Chem. 250, 56605668. 33. Harris, J. I. & Perham, R. N. (1968) Nature (London) 219, 1025-1028. 34. Davidson, B. E., Sajgo, M., Noller, H. G. & Harris, J. I. (1967) Nature (London) 216, 1181-1185. 35. Jones, G. M. T. & Harris, J. I. (1972) FEBS Lett. 22, 185-189. 36. Biesecher, G., Harris, J. I., Thierry, J. C., Walker, J. E. & Wonacott, A. J. (1977) Nature (London) 266,328-333. 37. Rossman, M. G., Lilias, A., Branden, C. I. & Banaszak, L. J. (1975) in The Enzymes, ed. Boyer, P. (Academic, New York), 3rd Ed., Vol. 11, pp. 61-102.
