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• W2000934302 abstract "The active site residue, His15, in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIAglucose, respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I Km for His15 → Asp HPr is increased 10-fold and Vmax is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp15 led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His15 → Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization. The possible involvement of Asn12 in an internal cyclization with Asp15 was eliminated by the Asn12 → Ala mutation in His15 → AspHPr. Asn12 substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the Nε2 atom of His15, but elimination of the hydrogen bond has only a 4-fold decrease inkcat/Km. The active site residue, His15, in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIAglucose, respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I Km for His15 → Asp HPr is increased 10-fold and Vmax is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp15 led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His15 → Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization. The possible involvement of Asn12 in an internal cyclization with Asp15 was eliminated by the Asn12 → Ala mutation in His15 → AspHPr. Asn12 substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the Nε2 atom of His15, but elimination of the hydrogen bond has only a 4-fold decrease inkcat/Km. Histidine-containing protein, HPr, is a small phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). 1The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; IEF, isoelectric focusing; PEP, phos phoenolpyruvate.1The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; IEF, isoelectric focusing; PEP, phos phoenolpyruvate. The PTS is a bacterial system that transports and phosphorylates many sugars and is involved in major regulatory events of carbohydrate metabolism (see reviews in Refs. 1Meadow N.D. Fox D.K. Roseman S. Annu. Rev. Biochem. 1990; 59: 497-542Crossref PubMed Scopus (302) Google Scholar, 2Postma P.W. Lengeler J.W. Jacobson G.R. Microbiol. Rev. 1993; 57: 543-594Crossref PubMed Google Scholar, 3Postma P.W. Lengeler J.W. Jacobson G.R. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D.C.1996Google Scholar, 4Lengeler J. Kahreis K. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics. 2. Elsevier Science Publishers B.V., Amsterdam1996: 573-598Google Scholar). HPr was first described by Kundig et al. (5Kundig W.W. Ghosh S. Roseman S. Proc. Natl. Acad. Sci. U. S. A. 1964; 52: 1067-1074Crossref PubMed Scopus (326) Google Scholar) when the function of accepting and donating a phosphoryl group from enzyme I to the sugar-specific enzymes II was established for Escherichia coli. These events result in phosphoprotein formation with a Nε2-P-histidine in both enzyme I (6Weigel N. Kukuruzinska M.A. Nakazawa T. Waygood E.B. Roseman S. J. Biol. Chem. 1982; 257: 14477-14491Abstract Full Text PDF PubMed Google Scholar) and the enzyme IIAsugar domains (7Meadow N.D. Roseman S. J. Biol. Chem. 1982; 257: 14526-14537Abstract Full Text PDF PubMed Google Scholar) and a more unstableN δ1-P-histidine in HPr (8Anderson B. Weigel N. Kundig W. Roseman S. J. Biol. Chem. 1971; 246: 7023-7033Abstract Full Text PDF PubMed Google Scholar, 9Waygood E.B. Erickson E.E. El-Kabbani O.A.L. Delbaere L.T.J. Biochemistry. 1985; 24: 6938-6945Crossref PubMed Scopus (41) Google Scholar). The structure of HPr from a number of species is now well established from both x-ray diffraction and NMR spectrometry approaches. The overall structure of the HPrs is described as an open-faced β-sandwich with a βαββαβα fold (see reviews in Refs. 10Jia Z. Quail J.W. Delbaere L.T.J. Waygood E.B. Biochem. Cell Biol. 1994; 72: 202-217Crossref PubMed Scopus (16) Google Scholar, 11Herzberg O. Klevit R. Curr. Opin. Struct. Biol. 1994; 4: 814-822Crossref PubMed Scopus (48) Google Scholar, 12Waygood E.B. Biochem. Cell Biol. 1998; 76: 359-367Crossref PubMed Scopus (18) Google Scholar). In order to help specify phosphoryl transfer to the Nδ1 atom of the His15 imidazole ring, various residues have been proposed to form hydrogen bonds with the Nε2 atom of the His15 imidazole. In E. coli, the involvement of a glutamate residue was suggested (9Waygood E.B. Erickson E.E. El-Kabbani O.A.L. Delbaere L.T.J. Biochemistry. 1985; 24: 6938-6945Crossref PubMed Scopus (41) Google Scholar) and was identified as Glu85 by the first 1The abbreviations used are: PTS, phosphoenolpyruvate:sugar phosphotransferase system; IEF, isoelectric focusing; PEP, phos phoenolpyruvate.H NMR structure (13Klevit R.E. Waygood E.B. Biochemistry. 1986; 25: 7774-7781Crossref PubMed Scopus (84) Google Scholar) and the 2.0 Å resolution x-ray structure (14Jia Z. Quail J.W. Waygood E.B. Delbaere L.T.J. J. Biol. Chem. 1993; 268: 22490-22501Abstract Full Text PDF PubMed Google Scholar), the latter showing that the interaction was with the C-terminal α-carboxylate. Replacement or deletion of Glu85 did not indicate a significant role for this residue (15Anderson J.W. Bhanot P. Georges F. Klevit R.E. Waygood E.B. Biochemistry. 1991; 30: 9601-9607Crossref PubMed Scopus (35) Google Scholar). NMR spectral properties of His15 inE. coli HPr were consistent with hydrogen bonding to the Nε2 atom (16van Dijk A.A. De Lange L.C.M. Bachovchin W.W. Robillard G.T. Biochemistry. 1990; 29: 8164-8171Crossref PubMed Scopus (51) Google Scholar), and van Nuland et al. (17van Nuland N.A.J. Boelens R. Scheek R.M. Robillard G.T. J. Mol. Biol. 1995; 246: 180-193Crossref PubMed Scopus (80) Google Scholar) suggested Asn12 as the most likely residue. Subsequently, in the 2.5 Å resolution structure of the complex of the Jel42 monoclonal antibody Fab fragment with HPr, the Asn12 side chain was found hydrogen-bonded to the Nε2 atom of His15 (18Prasad L. Waygood E.B. Lee J.S. Delbaere L.T.J. J. Mol. Biol. 1998; 280: 829-845Crossref PubMed Scopus (32) Google Scholar). Asn12 in E. coli HPr has been investigated because it is a site of deamidation, which occurs through the formation of a succinimide to form aspartate and isoaspartate at residue 12 (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar). The succinimide formation (Fig. 1A), especially at Asn-Gly pairs in a sequence leads to about 70% isoaspartic acid formation (20Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (296) Google Scholar, 21Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar, 22Wright H.T. Crit. Rev. Biochem. Mol. Biol. 1991; 27: 1-52Crossref Scopus (343) Google Scholar, 23Clarke S. Stephenson R.C. Lowenson J.D. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). This unusual amino acid can be repaired to aspartic acid by protein carboxylmethyltransferase (l-isoaspartate-(d-aspartate)O-methyltransferase) in peptides (24McFadden P.N. Clarke S. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2460-2464Crossref PubMed Scopus (167) Google Scholar), and the effective repair of residue 12 isoaspartic acid in HPr has been described (25Brennan T.V. Anderson J.W. Jia Z. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar). The substitution of aspartate or isoaspartate at residue 12 has modest effects on the phosphocarrier role of HPr (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar). The formation of succinimides can occur at aspartate residues but usually under different conditions than those that prevail for asparagine residues. A stable succinimide has been characterized in somatotropin where cyclization of an aspartyl residue under acidic conditions allows the isolation of the succinimide that is labile at alkaline pH (26Violand B.N. Schlittler M.R. Kolodziej E.W. Toren P.C. Cabonce M.A. Siegel N.R. Duffin K.L. Zobel J.F. Smith C.E. Tou J.S. Protein Sci. 1992; 1: 1634-1641Crossref PubMed Scopus (37) Google Scholar). A structure of a succinimide formed by an aspartyl residue at acidic pH has recently been described in lysozyme (27Noguchi S. Miyawaki K. Satow Y. J. Mol. Biol. 1998; 278: 231-238Crossref PubMed Scopus (42) Google Scholar). In both cases, the hydrolysis of the succinimide yielded both isoaspartic and aspartic acid. The mechanism of succinimide formation is not the only route by which deamidation can occur (20Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (296) Google Scholar, 21Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar, 22Wright H.T. Crit. Rev. Biochem. Mol. Biol. 1991; 27: 1-52Crossref Scopus (343) Google Scholar, 23Clarke S. Stephenson R.C. Lowenson J.D. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar), and among the possibilities is the formation of an isoimide shown in Fig. 1B. In this paper, we describe the effects of other substitutions at residue 12 and the lack of an absolute requirement for a histidine at residue 15 of HPr. Aspartate substitutes for histidine at residue 15 in phosphoryl acceptance and transfer, albeit inefficiently. In addition, the P-aspartyl residue leads to a spontaneous chemical rearrangement with the characteristics of catalyzed isoimide formation. Enzyme I, the enzymes IIsugar, and DNA oligonucleotides were obtained as described previously (28Anderson J.W. Pullen K. Georges F. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 12325-12333Abstract Full Text PDF PubMed Google Scholar). Ampholytes were from Amersham Pharmacia Biotech. DEAE paper (D81) was from Whatman. The Quik-Change site-directed mutagenesis kit was obtained from Stratagene. [32P]Phosphoenolpyruvate (PEP) was produced as described previously (29Mattoo R.L. Waygood E.B. Anal. Biochem. 1983; 128: 245-249Crossref PubMed Scopus (36) Google Scholar). His15 → Asp and His15→ Glu HPr mutants were produced by Dr. J. W. Anderson. Asn12 → Asp HPr has been described (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar), and all other mutations were produced by the Quik-Change site-directed mutagenesis kit according to the manufacturer's instructions. All mutations were in the ptsH gene incorporated into pUC19 (15Anderson J.W. Bhanot P. Georges F. Klevit R.E. Waygood E.B. Biochemistry. 1991; 30: 9601-9607Crossref PubMed Scopus (35) Google Scholar). His15 → Ala HPr was obtained from M Scholtz (Texas A & M). HPr and mutant HPrs were expressed in E. coli strain ESK108, which isptsH (28Anderson J.W. Pullen K. Georges F. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 12325-12333Abstract Full Text PDF PubMed Google Scholar), using the pUC(ptsH) plasmids with HPr expression under the control of its own promoter. Homogeneous protein was produced as described previously (15Anderson J.W. Bhanot P. Georges F. Klevit R.E. Waygood E.B. Biochemistry. 1991; 30: 9601-9607Crossref PubMed Scopus (35) Google Scholar). Yields were 50–500 mg of protein/30 g of wet weight of cells. His15 → Asp HPr (3 mg) was phosphorylated at 37 °C for 10 min in 10 mm potassium phosphate buffer, pH 7.0, with 5 mm PEP and 0.1 mg enzyme I. The three forms of His15 → Asp HPr, phosphorylated (lower pI), unphosphorylated, and cyclized (higher pI) were separated by anion exchange chromatography, Mini-Q-Sepharose column, and a Amersham Pharmacia Biotech Gradifrac system at 4 °C. The reaction mixture was loaded with 10 mm citrate-phosphate buffer, pH 4.6, and the column was eluted at 2 ml/min with a 20-ml gradient to 0.07 m NaCl in the same buffer. Fractions (0.5 ml) were collected, and protein elution was monitored at 214 nm. Protein sequencing was performed using an Applied Biosystems, Inc. model 471A sequencer equipped with a model MG5 microgradient pump and a blot cartridge for polyvinylidene difluoride-type membranes. Data were acquired and analyzed using an Applied Biosystems Inc. model 601A data system (30Robertson A.J. Ishikawa M. Gusta L.V. MacKenzie S.L. Plant Physiol. 1994; 105: 181-190Crossref PubMed Scopus (87) Google Scholar). The sequencing was carried out by Dr. S. Mackenzie (Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan, Canada). Mass spectrometry was performed using a Perseptive Biosystems Voyager ELITE matrix-assisted laser diode ionization-time of flight spectrometer at the Plant Biotechnology Institute. The samples were run in linear mode. Crystals of His15 → Asp HPr were grown by the hanging drop vapor diffusion method at 14 °C. Washing and seeding of microcrystals was used (31Thaller C. Weaver L.H. Eichele G. Wilson E. Karlsson R. Jansonius J.N. J. Mol. Biol. 1981; 147: 465-469Crossref PubMed Scopus (120) Google Scholar). Crystals formed in 0.1 m citrate phosphate buffer, pH 4.4, and 20–25% saturated ammonium sulfate. Crystals of the high pI form of His15 → Asp HPr, containing a putative cyclized Asp15 were grown similarly. Synchrotron diffraction data for His15 → Asp HPr were collected with a Brandeis CCD detector at the Brookhaven National Laboratory (Upton, NY). For the high pI form, data were collected at the Photon Factory (Tsukuba, Japan) using a wavelength of 1.0 Å and a screenless Weissenberg camera. The data were processed using DENZO and SCALEPACK (32.Otwinski, Z., Data Collection and Processing: Proceedings of the CCP4 Study Weekend, January 29–30, 1993, Warrington, UK, Sawyer, L., Isaacs, N., Bailey, S., 1993, 56, 62, SERC, Daresbury Laboratory, Warrington, UK.Google Scholar). The structures were solved as has previously been described for Ser46 → Asp HPr (33Napper S. Anderson J.W. Georges F. Quail J.W. Delbaere L.T.J. Waygood E.B. Biochemistry. 1996; 35: 11260-11267Crossref PubMed Scopus (31) Google Scholar) using the Amore suite of programs (34CCP4 Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar) with molecular replacement with wild type HPr (11Herzberg O. Klevit R. Curr. Opin. Struct. Biol. 1994; 4: 814-822Crossref PubMed Scopus (48) Google Scholar). Refinement was performed using the X-PLOR 3.1 package (35Brünger A.T. X-PLOR Manual, version 3.1. Yale University Press, New Haven, CT1993Google Scholar). The gene for enzyme IIAglc,crr, was isolated by polymerase chain reaction from pTSHIC9 (36Erni B. Zanolari B. Graff P. Kocher H.P. J. Biol. Chem. 1989; 264: 18733-18741Abstract Full Text PDF PubMed Google Scholar) and introduced into pT7-7 (37Studier F.W. Rosenberg A.H. Dunn J.H. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar) using the NdeI and BamHI restriction endonuclease sites. Enzyme IIglc was expressed in E. coli strain ESK262, which is Kan R::ptsH, following mid-log phase induction by 0.5 mmisopropylthiogalactoside. This strain was constructed by ligating the KanR gene from pUC4 into the PstI restriction endonuclease site in ptsH in pAB65 (38Lee L.G. Britton P. Parra F. Boronat A. Kornberg H.L. FEBS Lett. 1982; 149: 288-292Crossref PubMed Scopus (12) Google Scholar). The linearized plasmid was used to transform E. coli strain DPB271 (39Russell C.B. Thaler D.S. Dahlquist F.W. J. Bacteriol. 1989; 171: 2609-2613Crossref PubMed Google Scholar), which is recD, and a ptsH gene replacement derivative was selected by kanamycin resistance. This E. coli strain ESK150 had no HPr detectable by assay, phosphorylation, or immunoreactivity as determined by standard methods (40Waygood E.B. Reiche B. Hengstenberg W. Lee J.S. J. Bacteriol. 1987; 169: 2810-2818Crossref PubMed Google Scholar). E. coli strain was a derivative of strain BL21 plysS (37Studier F.W. Rosenberg A.H. Dunn J.H. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar), which was transduced with P1-phage grown on strain ESK150 to produce a Kan R ::ptsH strain ESK262. Enzyme I was also overproduced in this strain using a similar plasmid. 2S. Brokx, J. Taylor, F. Georges, and E. B. Waygood, submitted for publication. His15 → Asp HPr and derivatives were assayed for methyl accepting ability by incubation with S-adenosyl-l-[methyl-14C]methionine and l-isoaspartate-(d-aspartate)O-methyltransferase. Wild type HPr was used as a control. The assays were performed by J. D. Lowenson and S. Clarke (UCLA) as described previously (25Brennan T.V. Anderson J.W. Jia Z. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar). Standard methods have been described for characterization of HPr:protein determinations (15Anderson J.W. Bhanot P. Georges F. Klevit R.E. Waygood E.B. Biochemistry. 1991; 30: 9601-9607Crossref PubMed Scopus (35) Google Scholar, 28Anderson J.W. Pullen K. Georges F. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 12325-12333Abstract Full Text PDF PubMed Google Scholar), isoelectric focusing, (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar), SDS-polyacrylamide gel electrophoresis and autoradiography (42Waygood E.B. Mattoo R.L. Peri K.G. J. Cell. Biochem. 1984; 25: 139-159Crossref PubMed Scopus (47) Google Scholar), rates of phosphohydrolysis (9Waygood E.B. Erickson E.E. El-Kabbani O.A.L. Delbaere L.T.J. Biochemistry. 1985; 24: 6938-6945Crossref PubMed Scopus (41) Google Scholar), and enzyme I and enzyme IIAsugar assays (15Anderson J.W. Bhanot P. Georges F. Klevit R.E. Waygood E.B. Biochemistry. 1991; 30: 9601-9607Crossref PubMed Scopus (35) Google Scholar, 28Anderson J.W. Pullen K. Georges F. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 12325-12333Abstract Full Text PDF PubMed Google Scholar, 43Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar). The following substitutions for His15in E. coli HPr were made: alanine, asparagine, aspartate, cysteine, glutamate, glutamine, serine, threonine, and tyrosine. Except for His15 → Asp HPr, none of these mutations showed any detectable activity when tested for activity with enzyme I by: (a) a spectrophotometric assay for enzyme I activity (43Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar); (b) [32P]-protein labeling by PEP detected by SDS-polyacrylamide gel electrophoresis and autoradiography (42Waygood E.B. Mattoo R.L. Peri K.G. J. Cell. Biochem. 1984; 25: 139-159Crossref PubMed Scopus (47) Google Scholar); (c) a gel shift of a band on an isoelectric focusing (IEF) gel because of the introduction of the phosphoryl group (9Waygood E.B. Erickson E.E. El-Kabbani O.A.L. Delbaere L.T.J. Biochemistry. 1985; 24: 6938-6945Crossref PubMed Scopus (41) Google Scholar); and (d) in vivo complementation of the fermentation negative phenotype of the ptsH strain, E. coliESK108. His15 → Asp HPr, when incubated with PEP, enzyme I, and Mg2+, revealed two new species, one with a lower pI and another with a higher pI (Fig. 2,A and B). The formation of the high pI form was not efficient at room temperature (22 °C) but was readily detected at 37 °C (Fig. 2C). When [32P]PEP was used, the lower pI band was shown to contain [32P]phosphate by autoradiography; the higher pI band did not have a phosphoryl group (Fig. 2, D and E). Enzyme I phosphotransfer activity was measured using His15 → Asp HPr and was shown to have a Km of 66 μm for His15 → Asp HPr (wild type HPr Km 6 μm) and a Vmax that was 0.1% of that obtained with wild type HPr. The impairment of the enzyme I reaction was large, and thus much higher amounts of enzyme I were used, greater than 100-fold compared with equivalent experiments with wild type HPr. Assays of the enzymes IIsugar with His15 → Asp HPr would require impractical amounts of enzyme I to meet the requirements of independence of the enzyme II reaction from P-HPr generation (43Waygood E.B. Meadow N.D. Roseman S. Anal. Biochem. 1979; 95: 293-304Crossref PubMed Scopus (66) Google Scholar). For this reason, sugar phosphorylation was not measured. In the experiment described below, which showed enzyme IIAglc phosphorylation using [32P]PEP, the protein preparations required the purification of all the PTS proteins from strains of E. colithat did not produce HPr. When this was done, phosphorylation of enzyme IIAglc that was dependent upon the presence of His15 → Asp HPr could be shown (Fig. 3). To assess reactions with other enzymes IIAsugar, an in vivo approach was used. When His15 → Asp HPr was overproduced in vivo, it would not complement sugar fermentation in the ptsHstrain, E. coli ESK108. His15 → Asp HPr has a 10,000-fold impairment; Ser46 → Asp HPr is the next most impaired HPr described, ∼1000-fold, and its overproduction in vivo results in delayed fermentation (33Napper S. Anderson J.W. Georges F. Quail J.W. Delbaere L.T.J. Waygood E.B. Biochemistry. 1996; 35: 11260-11267Crossref PubMed Scopus (31) Google Scholar). His15 → Asp HPr is not effective in the overall function of the PTS. The pH stability of the different pI species was found by carrying out the phosphorylation reaction and then putting samples at different pHs and following the progress of the loss of species by IEF. These results showed that the high pI form was more stable at acidic pH and that the P-Asp15 HPr was more stable at alkaline pH. The deamidation events at Asn38 and Asn12 in HPr (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar, 25Brennan T.V. Anderson J.W. Jia Z. Waygood E.B. Clarke S. J. Biol. Chem. 1994; 269: 24586-24595Abstract Full Text PDF PubMed Google Scholar) suggested that the higher pI form might be a succinimide ring (Fig. 1). This form results in the loss of a water molecule from the protein, a net loss of 18 mass units. Moreover, when the succinimide hydrolyzes, the normal distribution of products is about 70% isoaspartyl and 30% aspartyl. The resulting HPr species have very similar pIs and have been distinguished by the detection of doublet bands on IEF gels (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar). In gels such as shown in Fig. 2, there was no indication of doublet bands for any of the pI species. No increase in protein carboxymethyl transferase methylating activity could be detected with His15 → Asp HPr or the derivatives following phosphorylation. The methylation reaction requires isoaspartyl residues. In addition, N-terminal sequencing of His15 → Asp HPr preparations and the derivatives obtained after dephosphorylation gave normal recoveries of an aspartyl residue at position 15. If an isoaspartyl residue forms, the sequencing reactions do not proceed through the isoaspartyl residue (22Wright H.T. Crit. Rev. Biochem. Mol. Biol. 1991; 27: 1-52Crossref Scopus (343) Google Scholar). The high pI form of His15 → Asp HPr was purified as described under “Experimental Procedures.” Mass spectroscopy showed a molecular species with 18 mass units less than His15 → Asp HPr (Fig. 4). The isolated high pI form was sequenced from the N terminus, and the sequencing did not proceed beyond residue 14. Incubation of the isolated high pI form at pH 9 led to reversion to the normal His15 → Asp HPr, and N-terminal sequencing identified only an aspartyl residue at position 15 with normal recoveries. When phosphorylation of the reverted form was carried out as described in Fig. 2, the appearance of the phosphorylated and higher pI species was the same (results not shown). These results confirm an unusual structure at residue 15 and the reversibility of the whole process. These findings are consistent with either a succinimide ring formation followed by a very constrained hydrolysis reaction to yield only aspartate or the formation of an isoimide from which hydrolysis would always yield an aspartyl residue (Fig. 1). Phosphohydrolysis of P-Asp HPr was investigated at several pHs. The comparisons with P-His HPr are given in Fig. 5. The structure of His15 → Asp HPr was determined as described under “Experimental Procedures.” Crystallographic parameters are shown in Table I. The 1.5 Å resolution structure of His15 → Asp HPr is essentially the same as the 2.0 Å resolution structure of wild type HPr (14Jia Z. Quail J.W. Waygood E.B. Delbaere L.T.J. J. Biol. Chem. 1993; 268: 22490-22501Abstract Full Text PDF PubMed Google Scholar). However, His15 → Asp HPr had the two differences found in both the 1.6 Å resolution structure of Ser46 → Asp HPr (33Napper S. Anderson J.W. Georges F. Quail J.W. Delbaere L.T.J. Waygood E.B. Biochemistry. 1996; 35: 11260-11267Crossref PubMed Scopus (31) Google Scholar) and the 2.5 Å resolution structure of wild type HPr bound to the Fab fragment of the HPr-specific monoclonal antibody Jel42 (18Prasad L. Waygood E.B. Lee J.S. Delbaere L.T.J. J. Mol. Biol. 1998; 280: 829-845Crossref PubMed Scopus (32) Google Scholar); neither the tight β-turn involving Asn12 nor any torsion angle strain at residue 16 was found. The Asp15 residue was well defined (Fig. 6A). One of the oxygen atoms of the Asp15 carboxyl group is in essentially the same position as the Nδ1 atom in the His15 imidazole ring of wild type; the relative distances between the position of the His15 Nδ1 atom in wild type and the positions of the two Asp15 carboxyl oxygen atoms in His15 → Asp HPr are 1.0 and 2.0 Å (Fig. 6B). The C-terminal carboxyl group of Glu85, which has been found hydrogen-bonded with the Nε2 atom of His15, is found in the same position in His15→ Asp HPr (Fig. 6B) as described in wild type and Ser46 → Asp HPrs (14Jia Z. Quail J.W. Waygood E.B. Delbaere L.T.J. J. Biol. Chem. 1993; 268: 22490-22501Abstract Full Text PDF PubMed Google Scholar, 33Napper S. Anderson J.W. Georges F. Quail J.W. Delbaere L.T.J. Waygood E.B. Biochemistry. 1996; 35: 11260-11267Crossref PubMed Scopus (31) Google Scholar), but no hydrogen bond is formed. The side chain of Asp15 is involved in no hydrogen bonds.Table IRefinement parametersHis15 → Asp HPrHigh pI speciesUnit cella = 25.37 Å, b = 45.34 Å, c = 27.62 Å, β = 104.0 °a = 25.92 Å, b = 45.97 Å, c = 27.22 Å, β = 104.2 °Space groupP21P21Resolution1.5 Å1.8 ÅWater molecules7243Final R19.1%20.0%Number of reflections77515670Completeness96.5%97.5%The accession numbers are: His15 → Asp HPr, 1cm3, and the high pI species, 1cm2. Open table in a new tab The accession numbers are: His15 → Asp HPr, 1cm3, and the high pI species, 1cm2. Formation of a succinimide or an isoimide have optimal main chain ψ angles and side chain χ1 dihedral angles: ψ = −120°, χ1 = +60°, and ψ = +60°, χ1 = +120°, respectively (20Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (296) Google Scholar). For the residue 15 in His15 → Asp HPr, these angles are ψ = −170°, χ1 = +61°, which are considerably closer to the ideal values for succinimide formation. In addition to this structure, the high pI form, which contained the putative cyclized form, was crystallized, and diffraction data were collected within 10 days. The unit cell and refinement parameters are very similar to the normal His15 → Asp HPr (TableII). Only a well defined aspartyl residue was found at residue 15 (Fig. 6A) and in essentially the same position as in the normal His15 → Asp HPr (Fig. 6C).Table IIKinetic parameters of Asn 12 substitutions in HPrHPrEnzyme I KmEnzyme I VmaxRelativekcat/Kmμm%%Wild type6100100Asn12 → Ala125025Asn12 → AspaResult from Sharma et al. (19).156025Asn12→ Ser66565Asn12 → Thr66565a Result from Sharma et al. (19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab A novel cyclic compound involving Asn12 and Asp15 was possible. To eliminate the potential involvement of Asn12 in the formation of the high pI form, the double mutant of HPr, His15 → Asp and Asn12 → Ala was made and purified. In wild type HPr, the Asn12 → Ala mutation causes a small impairment in the enzyme I reaction (Table II). In His15 → Asp HPr, the Asn12 → Ala mutation leads to no detectable change in the formation of P-Asp HPr or the high pI species, and the IEF gel is essentially the same as presented in Fig. 2. In order to follow more closely the events at position 15, the complications with respect to deamidation events at Asn12 and Asn38 were eliminated by creating the following two triple mutants Asn12 → Ala, His15 → Asp, and Asn38 → Ala, and Asn12 → Ala, His15 → Asn, and Asn38 → Ala. The events of either cyclization and/or deamidation were carried out at 60 °C and at pH 5.0, 7.0, 8.0, and 10.0 followed by separation of products on IEF gels as shown in Fig. 7. These gels show that there was no indication of either cyclization or deamidation even after 90 min at either pH 5.0 or pH 10.0, suggesting that the location at residue 15 has no unusual propensity to either cyclize or produce a succinimide specifically. Deamidation of Asn15 would have caused a band shift on the IEF gels, and none was observed even though the main chain ψ angle and side chain χ1 dihedral angle of Asp15 (and presumably Asn15) were near optimal for succinimide formation. The following mutants were made in wild type HPr: Asn12 → Ala, Asn12 → Ser, and Asn12 → Thr. Each was expressed and purified, and kinetic parameters for enzyme I were determined. The results are presented in Table II. The active site of HPr has two conserved residues, His15 and Arg17, and various investigations of the structure of HPr indicate that a residue with hydrogen bonding potential should be found at residue 12 in HPrs from several species (18Prasad L. Waygood E.B. Lee J.S. Delbaere L.T.J. J. Mol. Biol. 1998; 280: 829-845Crossref PubMed Scopus (32) Google Scholar, 19Sharma S. Hammen P.K. Anderson J.W. Leung A. Georges F. Hengstenberg W. Klevit R.E. Waygood E.B. J. Biol. Chem. 1993; 268: 17695-17704Abstract Full Text PDF PubMed Google Scholar, 44Herzberg O. Reddy P. Reizer J. Kapadia G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2499-2503Crossref PubMed Scopus (122) Google Scholar, 45Kalbitzer H.R. Hengstenberg W. Eur. J. Biochem. 1993; 216: 205-214Crossref PubMed Scopus (42) Google Scholar). Substitution of Asn12 in E. coli HPr by either serine or threonine has little effect on the phosphorylation of HPr by enzyme I. Serine is found in Bacillus subtilis HPr and threonine in Staphylococus aureus and Streptococcus faecalis HPrs. The removal of the hydrogen bonding potential by replacement with alanine results in only modest changes (Table II), very similar to that previously reported for Asn12 → Asp HPr. The approximately 4-fold change inkcat/Km will presumably affect the physiological efficiency of HPr, but the hydrogen bonding potential of residue 12 does not appear to be a major requirement for the mechanism of phosphoryl transfer. These modest changes in activity concur with the lack of direct evidence in NMR spectra for a hydrogen bond between His15 and residue 12 (45Kalbitzer H.R. Hengstenberg W. Eur. J. Biochem. 1993; 216: 205-214Crossref PubMed Scopus (42) Google Scholar, 46Wittekind M. Reizer J. Klevit R.E. Biochemistry. 1990; 29: 7191-7200Crossref PubMed Scopus (40) Google Scholar, 47Wittekind M. Rajagopal P. Branchini B. Reizer J. Saier Jr., M.H Klevit R.E. Protein Sci. 1992; 1: 1363-1376Crossref PubMed Scopus (63) Google Scholar, 48van Nuland N.A. van Dijk A.A. Dijkstra K. van Hoesel R. Scheek R.M. Robillard G.T. Eur. J. Biochem. 1992; 203: 483-491Crossref PubMed Scopus (30) Google Scholar, 50van Nuland N.A.J. Grötzinger J. Dijkstra K. Scheek R.M. Robillard G.T. Eur. J. Biochem. 1992; 210: 881-891Crossref PubMed Scopus (35) Google Scholar, 51van Nuland N.A.J. Hangyi I.W. van Schaik R.C. Berendsen H.J.C. van Gunsteren W.F. Scheek R.M. Robillard G.T. J. Mol. Biol. 1994; 237: 544-559Crossref PubMed Scopus (73) Google Scholar). In contrast to the flexibility of requirement at residue 12, it was expected that histidine would be an absolute requirement at residue 15. However, His15 → Asp can be phosphorylated, and donate phosphate to at least IIAglc but with much reduced efficiency. The aspartyl residue is a partial structural analogue of histidine as shown in Fig. 6; one of the carboxyl oxygen atoms is structurally equivalent to the Nδ1 atom in histidine. Phosphoryl transfer between P-histidines and acyl phosphates is well established. Acetate kinase, in which a γ-glutaminyl phosphate is formed (52Jones B.E. Rajagopal P. Klevit R.E. Protein Sci. 1997; 6: 2107-2119Crossref PubMed Scopus (30) Google Scholar), interacts with enzyme I to form a Nε2-P-histidine (53Fox D.K. Roseman S. J. Biol. Chem. 1986; 261: 13487-13497Abstract Full Text PDF PubMed Google Scholar, 54Fox D.K. Meadow N.D. Roseman S. J. Biol. Chem. 1986; 261: 13498-13503Abstract Full Text PDF PubMed Google Scholar). Reactions in chemotaxis involve transfers of phosphoryl groups between Nε2-P-histidine in CheA (41Hess J.F. Bourret R.B. Simon M.I. Nature. 1988; 336: 138-143Crossref Scopus (233) Google Scholar) and aspartyl phosphate in CheY (49Sanders D.A. Gillece-Castro B.L. Stock A.M. Burlingame A.L. Koshland Jr., D.E. J. Biol. Chem. 1989; 264: 21770-21778Abstract Full Text PDF PubMed Google Scholar), which is an example of two component sensor systems. An interesting aspect of the aspartyl substitution of His15is that phosphorylation catalyzes formation of a cyclized compound. Cyclization reactions to form succinimides are established for both asparagine and aspartate, and the production of isoaspartyl from the hydrolysis of succinimides is established (20Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (296) Google Scholar, 21Geiger T. Clarke S. J. Biol. Chem. 1987; 262: 785-794Abstract Full Text PDF PubMed Google Scholar, 22Wright H.T. Crit. Rev. Biochem. Mol. Biol. 1991; 27: 1-52Crossref Scopus (343) Google Scholar, 23Clarke S. Stephenson R.C. Lowenson J.D. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). Although isoaspartyl formation is favored, constraints in a protein structure might cause the formation of only aspartyl or only isoaspartyl. It has been proposed (20Clarke S. Int. J. Pept. Protein Res. 1987; 30: 808-821Crossref PubMed Scopus (296) Google Scholar, 23Clarke S. Stephenson R.C. Lowenson J.D. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar) that a second form of cyclization can occur to yield an isoimide (Fig. 1), and the subsequent hydrolysis of this yields only aspartyl residues. There is no indication that isoaspartyl acid is formed from the cyclization of Asp15, and the stimulation of cyclization by phosphorylation has been anticipated (23Clarke S. Stephenson R.C. Lowenson J.D. Ahern T.J. Manning M.C. Stability of Protein Pharmaceuticals: Chemical and Physical Pathways of Protein Degradation. Plenum Press, New York1992: 1-29Google Scholar). When either asparaginyl or aspartyl residues are at residue 15 in HPr, there is no indication of rapid cyclization in the absence of phosphorylation. This suggests that the phosphorylation of aspartate 15 is the pathway by which the cyclization to an isoimide is catalyzed. In wild type HPr, the phosphoryl group bound to the Nδ1atom of His15 has hydrogen-bonding interactions with the amide nitrogens of residues 16 and 17. As the one of the carboxyl oxygens of the aspartyl at residue 15 is an analogue of the Nδ1 atom, it is reasonable to assume that this is why phosphorylation occurs and that the same interactions between the phosphoryl group and the amide nitrogens of residues 16 and 17 occurs. This would mean that the phosphoryl group is hydrogen-bonded to the amide nitrogen of residue 16, which would be involved in succinimide formation. However, it is proposed that this interaction (Fig. 1C) leads to a concerted reaction, in which the phosphoryl group extracts the proton from the amide nitrogen of residue 16, with the consequence that the phosphoryl group is a better leaving group, and that the carbonyl of residue 16 is more reactive in attacking the carbonyl of residue 15 to form the isoimide. The complete lack of detection of significant amounts of isoaspartic acid and the ability to isolate a cyclic intermediate with 18 mass units less than His15 → Asp HPr are difficult to reconcile with the formation of a succinimide. It is suggested that the structure formed is an isoimide, which has not been found before in proteins. The following are thanked for contributions to this work: George Wong for help with protein purifications; Katherine Dixon, who made the first observation of phosphorylated His15 → Asp HPr phosphorylation during an undergraduate research project; Kim Napper and Joan Smallshaw produced E. coli strain ESK150 by gene replacement; James Talbot for the construction of E. coli strain ESK238; Jon Lowenson and Steve Clarke (UCLA) for the protein methylation assays; Sam Mackenzie (Plant Biotechnology Institute, National Research Council of Canada) for amino acid sequencing; Dr. M. Suzuki, The Photon Factory for help with synchrotron data collection; and Jeremy Lee for discussions on the formation of isoimides." @default.
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• W2000934302 title "The Aspartyl Replacement of the Active Site Histidine in Histidine-containing Protein, HPr, of the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Can Accept and Donate a Phosphoryl Group" @default.
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