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• W1995686191 abstract "Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an αββα complex in mesophilic microorganisms. The α-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated β-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in thetrp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA,trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar αββα complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a β2-homodimer that has a high catalytic efficiencyk cat/K mindolebecause of a very lowK mindole but does not bind to TrpA . These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles. Tryptophan synthase catalyzes the last two steps in the biosynthesis of the amino acid tryptophan. The enzyme is an αββα complex in mesophilic microorganisms. The α-subunit (TrpA) catalyzes the cleavage of indoleglycerol phosphate to glyceraldehyde 3-phosphate and indole, which is channeled to the active site of the associated β-subunit (TrpB1), where it reacts with serine to yield tryptophan. The TrpA and TrpB1 proteins are encoded by the adjacent trpA and trpB1 genes in thetrp operon. The genomes of many hyperthermophilic microorganisms, however, contain an additional trpB2 gene located outside of the trp operon. To reveal the properties and potential physiological role of TrpB2, the trpA,trpB1, and trpB2 genes of Thermotoga maritima were expressed heterologously in Escherichia coli, and the resulting proteins were purified and characterized. TrpA and TrpB1 form the familiar αββα complex, in which the two different subunits strongly activate each other. In contrast, TrpB2 forms a β2-homodimer that has a high catalytic efficiencyk cat/K mindolebecause of a very lowK mindole but does not bind to TrpA . These results suggest that TrpB2 acts as an indole rescue protein, which prevents the escape of this costly hydrophobic metabolite from the cell at the high growth temperatures of hyperthermophiles. phosphoribosyl anthranilate isomerase indoleglycerol phosphate synthase TrpB, α- and β-subunits of tryptophan synthase, respectively E. coli 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid glyceraldehyde 3-phosphate glyceraldehyde-3-phosphate dehydrogenase indoleglycerol phosphate indole pyridoxal 5′-phosphate T. maritima Hyperthermophilic microorganisms grow optimally close to the boiling point of water (1Stetter K.O. FEBS Lett. 1999; 452: 22-25Crossref PubMed Scopus (278) Google Scholar). It is interesting to identify those molecular adaptations that allow proper function of metabolism under these extreme conditions (2Daniel R.M. Cowan D.A. Cell. Mol. Life Sci. 2000; 57: 250-264Crossref PubMed Scopus (127) Google Scholar). In particular, enzymes from hyperthermophiles must be extremely thermostable, and labile metabolites must be protected from spontaneous degradation (3Jaenicke R. Böhm G. Curr. Opin. Struct. Biol. 1998; 8: 738-748Crossref PubMed Scopus (686) Google Scholar, 4Sterner R. Liebl W. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 39-106Crossref PubMed Scopus (325) Google Scholar, 5Vieille C. Zeikus G.J. Microbiol. Mol. Biol. Rev. 2001; 65: 1-43Crossref PubMed Scopus (1645) Google Scholar). The pathway of tryptophan biosynthesis from chorismate comprises seven catalytic functions (6Yanofsky C. Miles E. Bauerle R. Kirschner K. Creighton T.E. The Encyclopedia of Molecular Biology. 4. John Wiley & Sons, Inc., New York1999: 2276-2689Google Scholar). In most organisms, the trp genes are organized in operons, which guarantees their coordinated expression in response to the amount of tryptophan available in the growth medium (7Yanofsky C. Annu. Rev. Biochem. 2001; 70: 1-37Crossref PubMed Scopus (37) Google Scholar). The order of the trp operon from the hyperthermophileThermotoga maritima trpE(GD)CFBA (8Sterner R. Dahm A. Darimont B. Ivens A. Liebl W. Kirschner K. EMBO J. 1995; 14: 395-4404Crossref Scopus (39) Google Scholar) resembles the organization of the trp operon from Escherichia coli (9Yanofsky C. Platt T. Crawford I.P. Nichols B.P. Christie G.E. Horowitz H. VanCleeput M. Wu A.M. Nucleic Acids Res. 1981; 9: 6647-6668Crossref PubMed Scopus (219) Google Scholar). The last four steps of tryptophan biosynthesis are catalyzed by phosphoribosyl anthranilate isomerase (TrpF),1 indoleglycerol phosphate synthase (TrpC), and the α- and β-subunits of tryptophan synthase (TrpA and TrpB1, respectively). It was shown that TrpF and TrpC from T. maritima are far more thermostable than their homologs from mesophiles (10Sterner R. Kleeman G.R. Szadkowski H. Lustig A. Hennig M. Kirschner K. Protein Sci. 1996; 5: 2000-2008Crossref PubMed Scopus (76) Google Scholar, 11Merz A. Knöchel T. Jansonius J.N. Kirschner K. J. Mol. Biol. 1999; 288: 753-763Crossref PubMed Scopus (62) Google Scholar), probably because of an increased association state in the case of TrpF (12Hennig M. Sterner R. Kirschner K. Jansonius J.N. Biochemistry. 1997; 36: 6009-6016Crossref PubMed Scopus (89) Google Scholar, 13Thoma R. Hennig M. Sterner R. Kirschner K. Struct. Fold. Des. 2000; 8: 265-276Abstract Full Text Full Text PDF Scopus (89) Google Scholar) and an increased number of salt bridges in the case of TrpC (11Merz A. Knöchel T. Jansonius J.N. Kirschner K. J. Mol. Biol. 1999; 288: 753-763Crossref PubMed Scopus (62) Google Scholar, 14Knöchel T. Pappenberger A. Jansonius J.N. Kirschner K. J. Biol. Chem. 2002; 277: 8626-8634Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Moreover, both TrpF and TrpC from T. maritima are catalytically more active at 80 °C than the orthologous enzymes from E. coli at 37 °C, thus outrunning the unproductive hydrolysis of their thermolabile substrates under physiological conditions (10Sterner R. Kleeman G.R. Szadkowski H. Lustig A. Hennig M. Kirschner K. Protein Sci. 1996; 5: 2000-2008Crossref PubMed Scopus (76) Google Scholar, 11Merz A. Knöchel T. Jansonius J.N. Kirschner K. J. Mol. Biol. 1999; 288: 753-763Crossref PubMed Scopus (62) Google Scholar,15Sterner R. Merz A. Thoma R. Kirschner K. Methods Enzymol. 2001; 331: 270-280Crossref PubMed Scopus (3) Google Scholar). Less is known about the specific structural and functional adaptations of the tryptophan synthase from T. maritima, which catalyzes the conversion of indoleglycerol phosphate (IGP) and serine to tryptophan (16Dahm A. Molecular Evolution of Thermostable TIM-Barrel Enzymes of Tryptophan Biosynthesis. University of Basel, Switzerland1997Google Scholar). The tryptophan synthases characterized so far consist of two TrpA (α) and two TrpB1 (β) structural entities, which are organized either as four monofunctional subunits or as two bifunctional αβ-subunits (17Miles E.W. Adv. Enzymol. Rel. Areas. Mol. Biol. 1991; 64: 93-173PubMed Google Scholar). The x-ray structure at 2.8 Å resolution of the tryptophan synthase from Salmonella typhimurium revealed an αββα quaternary structure (18Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar). The structure of isolated TrpA from Pyrococcus furiosus confirmed that this enzyme has a (βα)8-barrel fold (19Yamagata Y. Ogasahara K. Hioki Y. Lee S.J. Nakagawa A. Nakamura H. Ishida M. Kuramitsu S. Yutani K.J. J. Biol. Chem. 2001; 276: 11062-11071Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), which is the most frequently encountered topology among single domain proteins and is also adopted by TrpF and TrpC (20Wierenga R.K. FEBS Lett. 2001; 492: 193-198Crossref PubMed Scopus (319) Google Scholar, 21Höcker B. Jürgens C. Wilmanns M. Sterner R. Curr. Opin. Biotechnol. 2001; 12: 376-381Crossref PubMed Scopus (79) Google Scholar). TrpB1 consists of two domains, which both comprise a central open β-sheet that is surrounded by α-helices (18Hyde C.C. Ahmed S.A. Padlan E.A. Miles E.W. Davies D.R. J. Biol. Chem. 1988; 263: 17857-17871Abstract Full Text PDF PubMed Google Scholar). TrpA catalyzes the aldolytic cleavage of IGP to glyceraldehyde 3-phosphate (GA3P) and indole, which condenses with serine at the active site of TrpB1 to yield tryptophan (Fig.1). The hydrophobic intermediate indole passes directly from the α-site to the β-site via a long tunnel, which prevents its loss from the cell by diffusion across the cytoplasmic membrane (22Miles E.W. Rhee S. Davies D.R. J. Biol. Chem. 1999; 274: 12193-12196Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 23Huang X. Holden H.M. Raushel F.M. Annu. Rev. Biochem. 2001; 70: 149-180Crossref PubMed Scopus (312) Google Scholar). There is pronounced allosteric communication between the TrpA and TrpB1 subunits from E. coli, which is reflected in a mutual activation of their catalytic activities which keeps the two reactions in phase and prevents accumulation of indole (24Pan P. Woehl E. Dunn M.F. Trends Biochem. Sci. 1997; 22: 22-27Abstract Full Text PDF PubMed Scopus (92) Google Scholar). It appears that the αββα complex is in an equilibrium between a low activity “open” and a high activity “closed” state, which is shifted by allosteric ligands and monovalent cations (25Fan Y.X. McPhie P. Miles E.W. Biochemistry. 2000; 39: 4692-4703Crossref PubMed Scopus (50) Google Scholar). The basis of the corresponding conformational transitions has been characterized by x-ray structure analysis of a number of enzyme-ligand complexes (26Rhee S. Parris K.D. Hyde C.C. Ahmed S.A. Miles E.W. Davies D.R. Biochemistry. 1997; 36: 7664-7680Crossref PubMed Scopus (143) Google Scholar, 27Rhee S. Miles E.W. Mozzarelli A. Davies D.R. Biochemistry. 1998; 37: 10653-10659Crossref PubMed Scopus (40) Google Scholar, 28Rhee S. Miles E.W. Davies D.R. J. Biol. Chem. 1998; 273: 8553-8555Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 29Schneider T.R. Gerhardt E. Lee M. Liang P.H. Anderson K.S. Schlichting I. Biochemistry. 1998; 37: 5394-5406Crossref PubMed Scopus (134) Google Scholar, 30Weyand M. Schlichting I. Biochemistry. 1999; 38: 16469-16480Crossref PubMed Scopus (77) Google Scholar). Recently, the genome sequencing of T. maritima (31Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. White O. Salzberg S.L. Smith H.O. Fraser C.M. Nature. 1999; 399: 323-329Crossref PubMed Scopus (1212) Google Scholar) and of other hyperthermophiles has identified a trpB2 gene outside of the trp operon. To reveal the roles in tryptophan biosynthesis of the two different TrpB variants, tmtrpA, tmtrpB1, and tmtrpB2 fromT. maritima were expressed heterologously in E. coli, and the corresponding protein products were purified and characterized by hydrodynamic measurements and steady-state enzyme kinetics. The results show that tmTrpB1 associates with tmTrpA to an αββα complex, in which the two different subunits strongly activate each other. tmTrpB2, which does not bind to tmTrpA but is catalytically highly active, has an extremely lowKm value for indole. It appears that tmTrpB1 has the same role in tryptophan biosynthesis as the known TrpB1 enzymes from mesophiles, whereas tmTrpB2 acts as a salvage protein that prevents the loss of indole at the physiological growth temperatures of hyperthermophiles. Preparation of DNA, amplification, extraction, digestion with restriction endonucleases, ligation, and sequencing were performed as described (32Beismann-Driemeyer S. Sterner R. J. Biol. Chem. 2001; 276: 20387-20396Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The genes tmtrpA and tmtrpB1 were amplified by PCR using the plasmid pDStmtrpAB (16Dahm A. Molecular Evolution of Thermostable TIM-Barrel Enzymes of Tryptophan Biosynthesis. University of Basel, Switzerland1997Google Scholar) as template. For amplification of tmtrpA, the oligonucleotides 5′-GGTTCATATGAAGGGCTTTATTGCATACATC-3′ with a NdeI site (in boldface type) and 5′-CGATGAATTCTCATTTTCCGAGGAGTTCTTTT-3′ with anEcoRI site (in boldface type) were used as 5′- and 3′-primers, respectively. The tmtrpB1 gene was amplified with the primers 5′-GGATCATATGAAAGGTTACTTCGGTCCTTA-3′ and 5′-CCGTGAATTCTCATCTTATCCTCTCCCTGACGTA-3′, again introducingNdeI and EcoRI sites (in boldface type). Using the two newly introduced restriction sites, the amplified DNA fragments were cloned into different pET vectors (Stratagene), yielding the plasmids pET21a-tmtrpA and pET24a-tmtrpB1. The gene tmtrpB2 was amplified by PCR using genomic DNA ofT. maritima as the template. The primers 5′-ACCGCATATGAGAATTGTTGTGAA-3′ and 5′-CAGGAATTCAAGCTTTCACACGTACGCTGT-3′ were used, introducingNdeI and HindIII sites, respectively (in boldface type). The amplified DNA fragment was cloned into the vector pET21a to yield the plasmid pET21a-tmtrpB2. All inserts were sequenced entirely to exclude inadvertent PCR mutations. Heterologous expression of tmtrpA was conducted in E. coli BL21(DE3) cells containing the plasmid pET21a-tmtrpA. For reasons unknown so far, tmtrpA (and tmtrpB1 and tmtrpB2) are being expressed in the absence of isopropyl-1-thio-β-d-galactopyranoside. Therefore, the cells were grown overnight at 37 °C in 1 liter of LB medium supplemented with 150 μg/ml ampicillin but without the addition of isopropyl-1-thio-β-d-galactopyranoside. The cell pellet resulting from centrifugation was resuspended in 10 mmpotassium phosphate buffer at pH 7.5 and lysed by sonification (Branson Sonifier W-250, 3 × 3 min, 50% pulse, 0 °C). According to SDS-PAGE, 90% of tmTrpA was found in the soluble fraction of the cell extract. 100 units of benzonase (Merck) was added to this fraction, which was then incubated for 1 h at 37 °C to degrade nucleic acids and subsequently for 20 min at 75 °C to denature the benzonase. The resulting suspension was centrifuged (Sorvall SS34, 13,000 rpm, 30 min, 4 °C), and the pellet, which contained heat-labile host proteins, was discarded. The supernatant was loaded on an anion exchange column (Mono Q, 2 × 11 cm, Amersham Biosciences, Inc.) that was equilibrated with 10 mmpotassium phosphate buffer, pH 7.5, at room temperature. The column was washed with 4 column volumes of equilibration buffer, and bound proteins were eluted with a linear gradient of 0–750 mmpotassium chloride at pH 7.5. tmTrpA eluted at 150–200 mmpotassium chloride, as judged from SDS-PAGE and conductivity measurements. Sufficiently pure fractions were pooled, concentrated using Centricon-10 devices (Millipore), and dialyzed against 10 mm potassium phosphate buffer, pH 7.5, containing 50 mm potassium chloride. The protein was shock frozen in liquid nitrogen at a concentration of 10 mg/ml. The purification yielded 50 mg of tmTrpA out of 1 liter of cell culture with a purity of about 99% as judged by SDS-PAGE. For expression of tmtrpB1 and tmtrpB2, E. coli BL21(DE3) cells containing the plasmids pET24a-tmtrpB1 and pET21a-tmtrpB2 were used. Cells were grown as described for tmtrpA, with the exceptions that kanamycin instead of ampicillin was added for maintenance of pET24a-tmtrpB1 and 20 °C instead of 37 °C was used, which increased the fraction of soluble protein to about 40% for tmTrpB1 and about 10% for tmTrpB2 (33Kopetzki E. Schumacher G. Buckel P. Mol. Gen. Genet. 1989; 216: 149-155Crossref PubMed Scopus (89) Google Scholar). Harvesting of cells, cell lysis, incubation with benzonase, heat precipitation of host proteins, and anion exchange chromatography were performed as described for tmTrpA, with the exception that the buffer solutions were supplemented with 40 μm pyridoxal 5′-phosphate (PLP). Both tmTrpB proteins eluted from the Mono Q column at about 150–200 mm potassium chloride. Fractions containing either tmTrpB protein were pooled, concentrated using Centricon-10 devices, and loaded on a gel filtration column (Superdex 75, Hiload 26/60, Amersham Biosciences, Inc.) equilibrated with 50 mm potassium phosphate at pH 7.5, containing 300 mm potassium chloride and 40 μm PLP. The tmTrpB proteins, which eluted with a purity above 95% as judged from SDS-PAGE, were shock frozen in liquid nitrogen at concentrations of 4 mg/ml (tmTrpB1) and 1.5 mg/ml (tmTrpB2). From 1 liter of cell culture, 16 mg of tmTrpB1 and 6 mg of tmTrpB2 were obtained. The tmgapdh gene cloned into the plasmid pKM1 was expressed using E. coli BL21(DE3) cells (34Pappenberger G. Schurig H. Jaenicke R. J. Mol. Biol. 1997; 274: 676-683Crossref PubMed Scopus (91) Google Scholar). The cells were grown at 37 °C in 1 liter of LB medium supplemented with 150 μg/ml ampicillin, inducted with 1 mmisopropyl-1-thio-β-d-galactopyranoside atA 600 = 0.6, and incubated overnight. Harvesting of cells, cell lysis, and incubation with benzonase were performed in a way similar to that described for tmTrpA, but a 50 mm EPPS buffer at pH 7.5 was used instead of 10 mm potassium phosphate. After heat precipitation of host proteins at 75 °C for 30 min, tmGAPDH was pure to 90%. The protein was dialyzed against 10 mm EPPS buffer at pH 7.5, containing 10 mm potassium chloride, concentrated to 5.2 mg/ml using Centricon-10 devices, and shock frozen in liquid nitrogen. From 1 liter of cell culture, 14 mg of tmGAPDH was obtained. Purification of proteins was followed by electrophoresis on 12.5% SDS-polyacrylamide gels using the system of Laemmli (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207031) Google Scholar) and staining with Coomassie Blue. The concentration of tmTrpA was determined using the molar extinction coefficient ε280 = 18.9 mm−1cm−1, which was calculated from the amino acid sequence (36Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3437) Google Scholar). The concentrations of tmTrpB1 and tmTrpB2 were determined according to Bradford (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215700) Google Scholar) because the strong absorption at 280 nm of the bound cofactor PLP impeded a reliable calculation of ε280 from the amino acid sequences. Analytical gel filtration was performed at a flow rate of 0.5 ml/min on a Superdex 75 column (1 × 30 cm) that was equilibrated with 50 mm potassium phosphate buffer at pH 7.5 at 25 °C containing 300 mm potassium chloride. Apparent molecular masses were calculated from the corresponding elution volumes, using a calibration curve that was obtained with standard proteins. Sedimentation velocity and sedimentation equilibrium runs were performed at 20 °C in a Beckman analytical ultracentrifuge (model Optima XLA), monitoring the absorption at 277 nm. The velocity runs were performed at 54,000 (tmTrpA) or 52,000 rpm (other proteins) and the equilibrium runs at 22,000 rpm (tmTrpA) or 8,000 rpm (other proteins). The proteins were dissolved in 100 mm potassium phosphate at pH 7.5, containing 180 mm potassium chloride and 40 μm PLP. For analysis of the equilibrium runs, a floating base-line computer program that adjusts the baseline absorbance (A) was used to obtain the best linear fit of lnAversus the square of the radial distance (r 2). Molecular masses were calculated assuming a partial specific volume of 0.73 ml/g. The cleavage of IGP to GA3P and indole (A-reaction; Fig. 1) was measured under steady-state conditions between 30 and 60 °C in a coupled enzymatic assay (38Creighton T.E. Eur. J. Biochem. 1970; 13: 1-10Crossref PubMed Scopus (85) Google Scholar). In this assay, arsenate and GA3P produced by tmTrpA were converted by tmGAPDH into 1-arseno-3-phosphoglycerate upon reduction of NAD+ to NADH. The reaction is irreversible because of the spontaneous hydrolysis of 1-arseno-3-phosphoglycerate into arsenate and 3-phosphoglycerate. Initial velocities were measured by absorption spectroscopy and analyzed using Δε340 (NADH − NAD+) = 6.22 mm−1cm−1. Initial velocities were measured, andV max andK mIGP were determined with a direct linear plot (39Eisenthal R. Cornish-Bowden A. Biochem. J. 1974; 139: 715-720Crossref PubMed Scopus (1237) Google Scholar). The conversion of indole and serine to tryptophan (B-reaction; Fig. 1) catalyzed by tmTrpB1 or tmTrpB2 was measured at 80 °C by absorption spectroscopy and analyzed using Δε290 (Trp − indole) = 1.89 mm−1 cm−1 (40Faeder E.J. Hammes G.G. Biochemistry. 1970; 9: 4043-4049Crossref PubMed Scopus (80) Google Scholar). Initial velocities were measured as a function of the concentration of either indole or serine, with the other substrate being present at saturating concentrations. In the case of tmTrpB2, entire progress curves at saturating concentrations of serine were analyzed with the integrated form of the Michaelis-Menten equation (41Hommel U. Eberhard M. Kirschner K. Biochemistry. 1995; 34: 5429-5439Crossref PubMed Scopus (51) Google Scholar), which allowed determination of the upper limit ofK mIND. The conversion of IGP and serine to tryptophan (AB-reaction; Fig. 1) catalyzed by [tmTrpA· tmTrpB1]2 between 30 and 60 °C was followed by absorption spectroscopy and analyzed using Δε290 (Trp − IGP) = 0.56 mm−1 cm−1 (42Brzovic P.S. Ngo K. Dunn M.F. Biochemistry. 1992; 31: 3831-3839Crossref PubMed Scopus (98) Google Scholar). Alternatively, the AB-reaction was followed in a coupled reaction, which is similar to that used to follow the A-reaction (43Lane A.N. Kirschner K. Biochemistry. 1991; 30: 479-484Crossref PubMed Scopus (63) Google Scholar). Initial velocities were measured as a function of the concentration of either IGP or serine, with the other substrate being present at saturating concentrations. The k cat and Km values of the A- and the AB-reactions determined between 30 and 60 °C were extrapolated to 80 °C by an Arrhenius plot. The genes trpA and trpB1 of the hyperthermophilic bacterium T. maritima are adjacent in thetrp operon (8Sterner R. Dahm A. Darimont B. Ivens A. Liebl W. Kirschner K. EMBO J. 1995; 14: 395-4404Crossref Scopus (39) Google Scholar). The sequencing of the whole genome ofT. maritima (31Nelson K.E. Clayton R.A. Gill S.R. Gwinn M.L. Dodson R.J. Haft D.H. Hickey E.K. Peterson J.D. Nelson W.C. Ketchum K.A. McDonald L. Utterback T.R. Malek J.A. Linher K.D. Garrett M.M. Stewart A.M. Cotton M.D. Pratt M.S. Phillips C.A. Richardson D. Heidelberg J. Sutton G.G. Fleischmann R.D. Eisen J.A. White O. Salzberg S.L. Smith H.O. Fraser C.M. Nature. 1999; 399: 323-329Crossref PubMed Scopus (1212) Google Scholar) identified a gene outside of thetrp operon, which has significant sequence similarity totrpB1 and was designated trpB2. It is likely that the trpB2 gene is expressed in T. maritimabecause the upstream region on the genome contains consensus sequences that are typical of bacterial promoters and ribosome binding sites (data not shown). A data base search revealed that trpB2 genes are also present in the genomes of most of the other investigated hyperthermophilic Bacteria and Archaea but are generally absent from the genomes of mesophiles. A phylogenetic tree based on amino acid sequence comparisons shows that TrpB1 and TrpB2 proteins form two separate groups (Fig. 2). Within the two groups, proteins display sequence identities of about 60%, whereas between members from different groups the identities are only about 30%. Most hyperthermophiles contain one TrpB1 and one TrpB2 protein; others, for example Sulfolobus solfataricus, possess two different TrpB2 variants but lack TrpB1. Fig. 3 a presents the amino acid sequence alignment of the two TrpB variants from T. maritima, tmTrpB1 and tmTrpB2, which show an overall identity of 38%. It is evident that those amino acids, which are conserved both in the TrpB1 and TrpB2 sequences, cluster at the putative active sites. In contrast, amino acids that are conserved in only one of the two TrpB groups are distributed along the sequences. The major differences between the two proteins are a long N-terminal extension and two shorter insertions in tmTrpB2, which are located in regions where tmTrpB1 interacts with tmTrpA, as deduced from the structure ofS. typhimurium tryptophan synthase (Fig. 3 b).Figure 3Similarities and differences between tmTrpB1 and tmTrpB2. Panel a, amino acid sequence alignment of tmTrpB1 and tmTrpB2 (excerpt from the multiple sequence alignment, on which the phylogenetic tree of Fig. 2 is based). The amino acids in black boxes are conserved in all TrpB1and TrpB2 sequences; the amino acids in blue andgreen boxes are conserved only in all TrpB1 or in all TrpB2 sequences, respectively. *, amino acids in a distance of less or equal to 4 Å from the nascent tryptophan in TrpB1 from S. typhimurium (PDB code 2TYS). Blue bars, amino acids corresponding to the communication (COMM) domain (29Schneider T.R. Gerhardt E. Lee M. Liang P.H. Anderson K.S. Schlichting I. Biochemistry. 1998; 37: 5394-5406Crossref PubMed Scopus (134) Google Scholar) in tmTrpB1; magenta bars, amino acids that are present only in tmTrpB2. Panel b, x-ray structure of the tryptophan synthase complex from S. typhimurium (stereo view). For clarity, only one α- and one β-subunit of the αββα complex are shown. Thetips of the magenta arrows mark the location of the N-terminal extension and the insertions within the COMMdomain (29Schneider T.R. Gerhardt E. Lee M. Liang P.H. Anderson K.S. Schlichting I. Biochemistry. 1998; 37: 5394-5406Crossref PubMed Scopus (134) Google Scholar) of tmTrpB2, which are not present in tmTrpB1 (seepanel a). Yellow substrates label the active sites, namely IGP bound to TrpA (PDB code 1QOQ) and the Trp·PLP complex bound to TrpB (PDB code 1TYS). The N and C termini of the β-subunit are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The tmtrpA, tmtrpB1, and tmtrpB2 genes were cloned into different pET vectors and expressed heterologously in E. coli BL21(DE3) cells (44Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6003) Google Scholar). tmTrpA could be produced in soluble form at 37 °C, but tmtrpB1 and tmtrpB2 had to be expressed at 20 °C to suppress in part the formation of insoluble aggregates (33Kopetzki E. Schumacher G. Buckel P. Mol. Gen. Genet. 1989; 216: 149-155Crossref PubMed Scopus (89) Google Scholar). The resulting thermostable tmTrpA, tmTrpB1, and tmTrpB2 proteins were purified from the soluble fraction of the cell extract, using a heat step to remove thermolabile host proteins followed by ion exchange chromatography. The three proteins were more than 95% pure, as judged from SDS-PAGE (data not shown). The [tmTrpA·tmTrpB1]2 complex was produced by mixing tmTrpA and tmTrpB1 (see below). Analytical gel filtration on a calibrated Superdex 75 HR column was used to test whether tmTrpB1 and tmTrpB2 form a complex with tmTrpA at 25 °C. The results are summarized in Fig.4. Separately, tmTrpA, tmTrpB1, and tmTrpB2 elute as well defined peaks. When tmTrpB1 is mixed with a molar excess of tmTrpA, the tmTrpB1 peak is replaced by a new and faster elution peak, which represents a complex of tmTrpA and tmTrpB1 (Fig.4 a). In contrast, the elution profile of a mixture of tmTrpA and tmTrpB2 excludes any significant complex formation between these proteins (Fig. 4 b). The elution time of separated tmTrpA corresponds to a molecular mass of 26.8 kDa, comparing well with the calculated molecular mass for the monomer (26.7 kDa). The elution times of tmTrpB1 and tmTrpB2, however, correspond to molecular masses of 49.4 and 61.7 kDa, respectively, which are between the calculated molecular masses for the respective monomers (42.9 and 46.4 kDa) and homodimers (85.8 and 92.8 kDa). Analytical ultracentrifugation was therefore performed to clarify the association states of tmTrpB1 and tmTrpB2 and to assess the stoichiometry of the complex between tmTrpA and tmTrpB1 (Table I). Sedimentation velocity runs showed that the separated proteins are homogeneous species, yieldings 20,w values of 2.8 for tmTrpA and 5.4 for both tmTrpB1 and tmTrpB2. The analysis of sedimentation equilibrium runs confirms that separated tmTrpA exists mainly as an α-monomer and shows that both tmTrpB1 and tmTrpB2 are β2-dimers. Runs that were performed with a mixture of tmTrpA and tmTrpB1 show that they form an αββα complex, as observed for other investigated tryptophan synthases (6Yanofsky C. Miles E. Bauerle R. Kirschner K. Creighton T.E. The Encyclopedia of Molecular Biology. 4. John Wiley & Sons, Inc., New York1999: 2276-2689Google Scholar, 45Tang X.F. Ezaki S. Atomi H. Imanaka T. Eur. J. Biochem. 2000; 267: 6369-6377Crossref PubMed Scopus (12) Google Scholar). In accordance with analytical gel filtration, analytical ultracentrifugation detected no complex formation between tmTrpA and tmTrpB2.Table IMolecular masses and sedimentation coefficients (s20,w) of tmTrpA, tmTrpB1, tmTrpB2, and their combinationsProtein(s)1-aBetween 3 and 18 μmprotein was dissolved in 100 mm potassium phosphate, pH 7.5, at 20 °C, containing 40 μm PLP and 180 mm KCl.Calculated molecular mass (for the given stoichiometry)Apparent molecular mass (from sedimenta" @default.
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• W1995686191 title "A Novel Tryptophan Synthase β-Subunit from the HyperthermophileThermotoga maritima" @default.
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