FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034144975> ?p ?o ?g. }

• W2034144975 endingPage "20396" @default.
• W2034144975 startingPage "20387" @default.
• W2034144975 abstract "Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF withN′-((5′-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction. Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF withN′-((5′-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction. glutamine amidotransferase 5-aminoimidazole-4-carboxamide ribotide imidazole glycerol phosphate glutaminase subunit of ImGP synthase HisH from E. coli and T. maritima, respectively synthase subunit of ImGP synthase HisF from E. coliand T. maritima, respectively N′-((5′-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide N′-((5′-phosphoribosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide polymerase chain reaction polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 1,4-piperazinediethanesulfonic acid During the synthesis of various biomolecules including amino acids, nucleotides, and coenzymes, the amido group of glutamine is transferred to a large variety of acceptor substrates by glutamine amidotransferases (GATases)1(1Zalkin H. Smith J.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 87-144PubMed Google Scholar, 2Massière F. Badet-Denisot M.A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar). GATases catalyze two separate reactions at two active sites that are either located on a single polypeptide chain or on different subunits. In the glutaminase reaction, glutamine is hydrolyzed to glutamate and ammonia, which in the synthase reaction is added to an acceptor substrate that is specific for each GATase. There are two classes of GATases that can be discriminated by catalytically essential residues in their glutaminase domains (1Zalkin H. Smith J.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 87-144PubMed Google Scholar, 3Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar). The key feature of class I GATases is the catalytic triad Cys-His-Glu. Recent x-ray structure determinations of three class I GATases,Escherichia coli carbamoyl phosphate synthase (4Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (307) Google Scholar, 5Thoden J.B. Miran S.G. Phillips J.C. Howard A.J. Raushel F.M. Holden H.M. Biochemistry. 1998; 37: 8825-8831Crossref PubMed Scopus (89) Google Scholar),E. coli GMP synthase (6Tesmer J.J. Klem T.J. Deras M.L. Davisson V.J. Smith J.L. Nat. Struct. Biol. 1996; 3: 74-86Crossref PubMed Scopus (211) Google Scholar), and S. solfataricusanthranilate synthase (7Knöchel T. Ivens A. Hester G. Gonzalez A. Bauerle R. Wilmanns M. Kirschner K. Jansonius J.N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9479-9484Crossref PubMed Scopus (105) Google Scholar) indicate a common fold of their glutaminase domains, which is similar to the well known α/β hydrolase fold (8Ollis D.L. Cheah E. Cygler M. Dijkstra B. Frolow F. Franken S.M. Harel M. Remington S.J. Silman I. Schrag J. Protein Eng. 1992; 5: 197-211Crossref PubMed Scopus (1852) Google Scholar). Class II GATases belong to the large family of Ntn hydrolases (9Brannigan J.A. Dodson G. Duggleby H.J. Moody P.C. Smith J.L. Tomchick D.R. Murzin A.G. Nature. 1995; 378: 416-419Crossref PubMed Scopus (547) Google Scholar), and their only catalytically essential amino acid is the conserved N-terminal cysteine. The corresponding synthase domains within each class are structurally, evolutionary, and functionally unrelated (2Massière F. Badet-Denisot M.A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar), supporting the hypothesis that glutamine-hydrolyzing enzymes were recruited independently by previously ammonium-dependent enzymes (3Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar). Along these lines, most GATases can use ammonium salts as an alternative source of ammonia (1Zalkin H. Smith J.L. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 87-144PubMed Google Scholar, 2Massière F. Badet-Denisot M.A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar). The imidazole glycerol phosphate (ImGP) synthase is a class I GATase, which in bacteria constitutes a bienzyme complex consisting of the glutaminase subunit HisH and the synthase subunit HisF (10Alifano P. Fani R. Liò P. Lazcano A. Bazzicalupo M. Carlomagno M.S. Bruni C.B. Microbiol. Rev. 1996; 60: 44-69Crossref PubMed Google Scholar, 11Klem T.J. Davisson V.J. Biochemistry. 1993; 32: 5177-5186Crossref PubMed Scopus (97) Google Scholar). The ammonia produced by HisH reacts with the substrate of HisF, which isN′-((5′-phosphoribulosyl) formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR). The products of this reaction, ImGP and 5- aminoimidazole-4-carboxamide ribotide (AICAR), are further used in histidine and de novo purine biosynthesis, respectively. In yeast, the glutaminase and synthase activities are located on a single polypeptide chain, which is termed HIS7 (12Kuenzler M. Balmelli T. Egli C.M. Paravicini G. Braus G.H. J. Bacteriol. 1993; 175: 5548-5558Crossref PubMed Google Scholar, 13Chittur S.V. Chen Y. Davisson V.J. Protein Expression Purif. 2000; 18: 366-377Crossref PubMed Scopus (24) Google Scholar). The mechanism of glutamine hydrolysis by HisH can be deduced from the class I GATase carbamoyl phosphate synthase (5Thoden J.B. Miran S.G. Phillips J.C. Howard A.J. Raushel F.M. Holden H.M. Biochemistry. 1998; 37: 8825-8831Crossref PubMed Scopus (89) Google Scholar). However, due to the lack of a high resolution structure for the bienzyme complex, the ammonia transfer from HisH to HisF and the mechanism of the HisF reaction are only poorly understood. In order to address these questions, the thermostable variants tHisF and tHisH from Thermotoga maritima were produced in E. coli, purified and characterized by hydrodynamic and spectroscopic measurements, limited proteolysis, and steady-state enzyme kinetics. Moreover, the high resolution x-ray structure of isolated tHisF (14Lang D. Thoma R. Henn-Sax M. Sterner R. Wilmanns M. Science. 2000; 289: 1546-1550Crossref PubMed Scopus (264) Google Scholar) was used to identify and probe amino acid residues that are potentially involved in catalysis of the synthase reaction. It was shown that tHisH is activated by complex formation with tHisF containing ImGP or a substrate analogue bound to its active site. A flexible loop region in tHisF appears to be important for these functional interactions with tHisH. Furthermore, two aspartate residues at the active site of tHisF were demonstrated to be essential for catalysis. One of them was partially replaceable by glutamate, as shown by saturation random mutagenesis and complementation in vivo of an E. coli hisF deletion strain. Based on these findings, a chemically plausible mechanism for the HisF synthase reaction was derived, which involves general acid/base catalysis. Preparation of DNA, digestion with restriction endonucleases, and DNA ligation were performed as described (15Sambrook J. Fritsch E.E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Oligonucleotides were purchased from Metabion. DNA was amplified by PCR using cloned Pfupolymerase (Stratagene). For PCR with mutagenic oligonucleotides,Taq polymerase (Roche Diagnostics) was used. DNA was extracted from agarose gels using the QIAquick gel extraction kit (Qiagen). DNA sequencing was performed by the “Göttingen Genomics Laboratory” and by the “Zentrum für Molekulare Medizin der Universität Köln,” using standard methods. N-terminal protein sequencing was performed by Dr. Paul Jenö(Biozentrum, University of Basel) and the “Zentrum für Molekulare Medizin der Universität Köln,” again using standard methods. ThehisF gene variants from Thermotoga maritima(thisF; Ref. 16Thoma R. Schwander M. Liebl W. Kirschner K. Sterner R. Extremophiles. 1998; 2: 379-389Crossref PubMed Scopus (22) Google Scholar) encoding the synthase subunit of imidazole glycerol phosphate synthase were cloned into the expression vector pET11c (Novagen) using the restriction enzymes NdeI andBamHI. The hisH gene of T. maritima(thisH; Ref. 16Thoma R. Schwander M. Liebl W. Kirschner K. Sterner R. Extremophiles. 1998; 2: 379-389Crossref PubMed Scopus (22) Google Scholar) was amplified by PCR, using purified chromosomal DNA (Qbiogen) as a template. The oligonucleotides 5′-GGT GTG ATA GCA TGC GTA TCG-3′ with aSphI-site (in boldface type) and 5′-CTA CCA AGC TTC TGA AGA GAT CTA TCG-3′ with aHindIII-site (in boldface type) were used as 5′- and 3′-primers, respectively. Using the two newly introduced restriction sites, the amplified DNA fragment was cloned into the vector pDS56/RBSII/SphI (17Stüber D. Matile H. Garotta G. Lefkovits I. Pernis B. Immunological Methods. 4. Academic Press, Inc., Orlando, FL1990: 121-152Crossref Google Scholar) to yield the plasmid pDS56/RBSIISphI-thisH. All inserts were entirely sequenced to exclude inadvertent PCR mutations. Point mutations were introduced into thisF by PCR-based methods using either mutagenic 5′-primer for construction of thisF_C9A, D11N, K19S, or the megaprimer method (18Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar) for construction of thisF_D51N, N103A, D130N, D176N, and D183N. In both approaches, the plasmid pET11c-thisF (19Thoma R. Obmolova G. Lang D.A. Schwander M. Jeno P. Sterner R. Wilmanns M. FEBS Lett. 1999; 454: 1-6Crossref PubMed Scopus (33) Google Scholar) was used as the template. The following mutagenic 5′-primers were used (NdeI restriction sites are in boldface type, and base substitutions to introduce an amino acid exchange are underlined): 5′-TGA TGA AGACAT ATG CTC GCT AAA AGA ATA ATC GCG GCT CTC GAT-3′ for construction of C9A, 5′-TGA TGA AGA CAT ATG CTC GCT AAA AGA ATA ATC GCG TGC CTC AAT GTG AAA GAC-3′ for construction of D11N, and 5′-TGA TGA AGA CAT ATG CTC GCT AAA AGA ATA ATC GCG TGT CTC GAT GTG AAA GAC GGT CGT GTG GTG AGCGGA ACG AAC TTC-3′ for construction of K19S. In all PCRs, the oligonucleotide 5′-CCG GAT CCA GCG TCA TCA CAA-3′ containing a BamHI restriction site (in boldface type) was used as the 3′-primer. For the production of megaprimers, the following mutagenic oligonucleotides were used (base substitutions to introduce restriction sites for the control of the reaction are in boldface type, and base substitutions to introduce an amino acid exchange are underlined): 5′-CGC GGT A AT AT T CAG AAA AAC GAG-3′ with a new SspI restriction site for construction of D51N, 5′-CAC AGC CGC AGT GGC TAT GCT CAC CTT GTC-3′ with a new TspRI site for construction of N103A, 5′-CAC TCT TTT TGC ATTAAT CGC CAC GAC-3′ with a newVspI restriction site for construction of D130N, 5′-GAC AGAAAC GGC ACC AAA TCG G-3′ with a newBshNI restriction site for construction of D176N, and 5′-GGC ACA AAA TCG GGT TAC AACACT GAG ATG ATA AGG-3′ with a new TspRI restriction site for construction of D183N. For production of the megaprimers for thisF_D51N, N103A, and D130N, the corresponding mutagenic oligonucleotides listed above were used as 3′-primers, and the oligonucleotide 5′-TGA TGA AGACAT ATG CTC GCT AAA AG-3′ (NdeI restriction site in boldface type) was used as 5′-primer. The megaprimers were purified by agarose gel electrophoresis and used in the second PCR as 5′-primers, whereas the oligonucleotide 5′-CCG GAT CCA GCG TCA TCA CAA-3′ (BamHI restriction site in boldface type) was used as 3′-primer. The construction of the variants thisF_D176N and D183N followed the same protocol except that in the first PCR the mutagenic oligonucleotides were used as 5′-primers and the oligonucleotide 5′-CCG GAT CCA GCG TCA TCA CAA-3′ (BamHI restriction site in boldface type) was used as 3′-primer. The purified megaprimers were used as 3′-primers, and the oligonucleotide 5′-TGA TGA AGA CAT ATG CTC GCT AAA AG-3′ (NdeI restriction site in boldface type) was used as 5′-primer. The resulting full-length products were digested withNdeI and BamHI and ligated into pET11c. In order to confirm the base substitutions and to exclude inadvertent additional ones, all thisF gene variants were entirely sequenced. The thisF codons representing amino acids 11 and 130 were randomized in PCR-based approaches using degenerated primers and pET11c-thisF as template. For randomization of position 11, the oligonucleotide 5′-TAT ACG CAT GCT CGC TAA AAG AAT AAT CGC GTG CCT CNNSGT GAA GAC-3′ with a SphI restriction site (in boldface type) was used, where N represents equal molar mixtures of all four bases, and S represents an equal molar mixture of G and C. In a PCR, the degenerate oligonucleotide was used as 5′-primer, and the oligonucleotide 5′-GTC GAC GGA TCC ACA ACC CCT CCA G-3′ with a BamHI restriction site (in boldface type) was used as 3′-primer to yield an ensemble of thisF_D11NNS gene variants. For randomization of position 130, a megaprimer was produced using the degenerate oligonucleotide 5′-GTC GTG GCG ATT NNS GCA AAA AGA-3′ as 3′-primer and the oligonucleotide 5′-TAT ACG CAT GCT CGC TAA AAG AAT AAT CGC-3′ with a SphI restriction site (in boldface type) as 5′-primer. In a second PCR, the purified megaprimer was used as 5′-primer, and the oligonucleotide 5′-GTC GAC GGA TCC ACA ACC CCT CCA G-3′ with a BamHI restriction site (in boldface type) was used as 3′-primer to yield an ensemble of thisF_D130NNS gene variants. The PCR-amplified ensembles of thisF genes containing randomized codons at amino acid position 11 or 130 were ligated into a modified pDS56/RBSII vector (termed pTNA), which contains a truncated derivative of the tryptophanase operon promotor (20Merz A. Yee M.C. Szadkowski H. Pappenberger G. Crameri A. Stemmer W.P. Yanofsky C. Kirschner K. Biochemistry. 2000; 39: 880-889Crossref PubMed Scopus (108) Google Scholar) that permits constitutive gene expression in E. coli. The auxotrophic E. colistrain UTH860 (ΔhisF), which carries a mutant ehisF gene that encodes an inactive HisF protein (21Goldschmidt E.P. Cater M.S. Matney T.S. Butler M.A. Greene A. Genetics. 1970; 66: 219-229Crossref PubMed Google Scholar), was transformed separately with the two plasmid libraries and plated onto LB medium containing 150 μg ml−1 ampicillin (15Sambrook J. Fritsch E.E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The resulting lawns of at least 105 clones were rinsed off the plates, and plasmid DNA was prepared from the mixture of colonies. The ΔhisF strain was retransformed with these plasmid libraries, and aliquots were streaked on LB medium plates (nonselective plates) or on minimal medium plates (22Vogel H.J. Bonner D.M. J. Biol. Chem. 1956; 218: 97-106Abstract Full Text PDF PubMed Google Scholar) without histidine (selective plates) and incubated at 37 °C. From the nonselective plates, 16 colonies with randomized thisF codon 11 and 18 colonies with randomized thisF codon 130 were picked, and the corresponding thisF genes were entirely sequenced. The mutational saturation is given by the following,p≤1−1−∑i=1mfinEquation 1 where p is the probability that, form randomized codons appearing with the relative frequencyfi, practically all possible amino acid combinations are present in a library containing n independent clones (23Darimont B. Stehlin C. Szadkowski H. Kirschner K. Protein Sci. 1998; 7: 1221-1232Crossref PubMed Scopus (24) Google Scholar). Under the assumption that all 32 permitted codons appear with equal frequency (fi =132), mutational saturation (p ≥ 0.99) of one codon (m= 1) is attained with a library of more than 145 independent clones. Although the bases within codons 11 and 130 were not randomly distributed, every allowed base was found at all three codon positions. None of the thisF genes contained additional mutations. Considering the size of the two libraries, each of which contained at least 105 independent clones, it can be concluded that all 20 amino acids were represented at both randomized positions. To select for functional amino acids at position 11 or 130 of tHisF, for each library one selective plate was incubated for various time periods. A number of clones appeared on both plates overnight and, after 48 h, additional colonies appeared on the selective plate with thisF genes that were randomized at position 130. Incubation was continued for 1 week, but no additional colonies appeared. A number of colonies that grew on selective medium after different periods of time were picked, and the thisF_D11NNS or thisF_D130NNS genes that encoded functional tHisF proteins were sequenced. Wild-type tHisF and its variants containing individual amino acid exchanges were purified as described (19Thoma R. Obmolova G. Lang D.A. Schwander M. Jeno P. Sterner R. Wilmanns M. FEBS Lett. 1999; 454: 1-6Crossref PubMed Scopus (33) Google Scholar). The yield was between 7 and 17 mg of purified enzyme per g of wet cell mass. Heterologous expression of thisH was conducted in E. coli W3110 ΔtrpEA2 cells containing pDS56/ RBSIISphI-thisH and the repressor plasmid pDMI, 1, as described for hisA fromT. maritima (19Thoma R. Obmolova G. Lang D.A. Schwander M. Jeno P. Sterner R. Wilmanns M. FEBS Lett. 1999; 454: 1-6Crossref PubMed Scopus (33) Google Scholar). The cells were grown in 1 liter of LB medium supplemented with 0.15 mg/ml ampicillin and 0.075 mg/ml kanamycin. Overexpression of thisH was induced by adding 1 mm isopropyl-1-thio-β-d-galactopyranoside at an optical density at 600 nm of about 0.6, and incubation was continued overnight. The cell suspension was washed with 100 mmpotassium phosphate buffer at pH 7.5, containing 1 mm EDTA and 1 mm dithiothreitol, resuspended (5 ml of buffer per g wet cell mass), and lysed by sonification (Branson Sonifier W-250, 2 × 2 min, 50% pulse, 0 °C). According to SDS-PAGE, about 60% of tHisH were found in the soluble fraction of the cell extract. Benzonase (Merck) (50 units) was added to this fraction, which was then incubated for 1 h at 37 °C and subsequently for 20 min at 75 °C. The resulting suspension was centrifuged (Sorvall SS34, 12,000 rpm, 30 min, 4 °C), and the pellet, which contained heat-labile host proteins, was discarded. The supernatant was dialyzed against 10 mm Tris/HCl buffer at pH 8.0, containing 1 mm EDTA and 1 mm dithiothreitol, and loaded on an anion exchange column (POROS HQ20; 1 × 10 cm, PE Biosystems) that was equilibrated with the same buffer at room temperature. The column was washed with four volumes of equilibration buffer, and bound proteins were eluted with 1.5 liters of a linear gradient of 0–1m sodium chloride at pH 8.0. tHisH eluted between 130 and 150 mm sodium chloride, as judged from SDS-PAGE and conductivity measurements. Fractions containing tHisH were pooled, dialyzed against 10 mm potassium phosphate buffer at pH 7.5, containing 1 mm dithiothreitol, loaded on a hydroxylapatite column (3.6 × 20 cm; Novartis) that was equilibrated with the same buffer, and eluted with 2 liters of a linear gradient of 10–500 mm potassium phosphate. tHisH eluted between 100 and 150 mm potassium phosphate with a purity above 95%, as judged from SDS-PAGE. The purification yielded ∼5 mg of tHisH per g of wet cells. Fractions containing pure tHisH were pooled, dialyzed against 50 mm potassium phosphate buffer at pH 7.5, containing 1 mm EDTA and 1 mmdithiothreitol, concentrated to 1.8 mg/ml using Centricon-10 concentration devices (Millipore), and shock-frozen in liquid nitrogen. Purification of proteins was followed by electrophoresis on 12.5 or 15% SDS-polyacrylamide gels using the system of Laemmli (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and staining with Coomassie Blue. During purification, the protein concentration was determined according to Bradford (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). The concentration of purified proteins was determined with molar extinction coefficients at 280 nm that were calculated from the amino acid sequence (26Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). Analytical gel filtration was performed at a flow rate of 0.5 ml/min on a Superdex 75 column (1 × 30 cm; Amersham Pharmacia Biotech) that was equilibrated with 50 mm potassium phosphate at pH 7.5, containing 300 mm sodium chloride. Apparent molecular masses were determined from the corresponding elution volumes, using a calibration curve that was obtained with standard proteins. Sedimentation equilibrium runs were performed in a Beckman analytical ultracentrifuge (model Optima XLA), following the absorption at 278 nm. Runs with tHisF were performed as described (27Höcker B. Beismann-Driemeyer S. Hettwer S. Lustig A. Sterner R. Nat. Struct. Biol. 2001; 8: 32-36Crossref PubMed Scopus (131) Google Scholar). Runs with tHisH were performed at 24,000 rpm and protein concentrations of 12 and 23 μm in 50 mm potassium phosphate, pH 7.5, at 20 °C, containing 25 mm potassium chloride. Runs with tHisH-tHisF were performed at 18,000 and 24,000 rpm with 20 μm protein in 50 mm potassium phosphate at pH 7.5 at 20 °C, containing 300 mm sodium chloride. To determine apparent molecular masses, the runs were analyzed as described (27Höcker B. Beismann-Driemeyer S. Hettwer S. Lustig A. Sterner R. Nat. Struct. Biol. 2001; 8: 32-36Crossref PubMed Scopus (131) Google Scholar). Fluorescence spectra were recorded with a F-4500 spectrofluorimeter (Hitachi) or a Cary Eclipse spectrofluorimeter (Varian). Proteolytic stability was tested at room temperature by incubating 10 nmol of substrate protein with 64 pmol of trypsin in 1 ml of 50 mm potassium phosphate, pH 7.5. The reaction was stopped after different time intervals by adding one volume of 2× SDS-PAGE sample buffer and heating for 5 min at 95 °C. The time course of proteolysis was followed on Tris-Tricine gels containing 20% acrylamide (28Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). The ammonia-dependent activity of isolated tHisF was measured by recording entire progress curves in 50 mm Tris acetate buffer, pH 8.5, at 25 °C. In order to determineKmPRFAR, the enzyme was saturated with ammonia by adding 100 mm ammonium acetate corresponding to 14.4 mm NH3 at pH 8.5. PRFAR, 20 μm, were synthesized in situ from ProFAR, using a molar excess of HisA from T. maritima (19Thoma R. Obmolova G. Lang D.A. Schwander M. Jeno P. Sterner R. Wilmanns M. FEBS Lett. 1999; 454: 1-6Crossref PubMed Scopus (33) Google Scholar) and completely converted by tHisF to ImGP and AICAR. The reaction was quantified by the decrease in absorption at 300 nm, using Δε300(PRFAR-AICAR) = 5.64 mm−1 cm−1(11Klem T.J. Davisson V.J. Biochemistry. 1993; 32: 5177-5186Crossref PubMed Scopus (97) Google Scholar). In order to determine KmNH3, the reaction was performed in the presence of 50 μm PRFAR at various concentrations of ammonium acetate between 0 and 200 mm, corresponding to 0 and 35 mmNH3 at pH 8.5. The glutamine-dependent activity of the tHisH-tHisF complex was measured in an analogous way as the ammonia-dependent reaction of tHisF, but the pH value was set to 8.0. In order to determineKmPRFAR, the reaction was performed with 20 μm ProFAR and 5 mml-glutamine. To determineKmGln, the reaction was performed in the presence of 50 μm PRFAR at various concentrations of glutamine between 0 and 7 mm. The progress curves were analyzed with the integrated form of the Michaelis-Menten equation (29Hommel U. Eberhard M. Kirschner K. Biochemistry. 1995; 34: 5429-5439Crossref PubMed Scopus (51) Google Scholar), yielding values for Km andVmax. The glutaminase activity of tHisH in complex with liganded tHisF was measured in a coupled enzymatic assay with bovine liver glutamate dehydrogenase (Sigma). Glutamate that was produced by tHisH was oxidized by a molar excess of glutamate dehydrogenase in the presence of NAD+, yielding 2-oxoglutarate and NADH + H+ + NH4+. The reaction was quantified by the increase in absorption at 340 nm, using Δε340(NADH-NAD+) = 6300m−1 cm−1. The values for KmGln andVmax were deduced from initial velocity measurement at various glutamine concentrations. tHisF was produced and purified as described previously (19Thoma R. Obmolova G. Lang D.A. Schwander M. Jeno P. Sterner R. Wilmanns M. FEBS Lett. 1999; 454: 1-6Crossref PubMed Scopus (33) Google Scholar). The thisH gene (16Thoma R. Schwander M. Liebl W. Kirschner K. Sterner R. Extremophiles. 1998; 2: 379-389Crossref PubMed Scopus (22) Google Scholar) was cloned into the expression vector pDS56/RBSII/SphI. Proteins were expressed in E. coli from this plasmid under the control of a lacoperator system (17Stüber D. Matile H. Garotta G. Lefkovits I. Pernis B. Immunological Methods. 4. Academic Press, Inc., Orlando, FL1990: 121-152Crossref Google Scholar). tHisH was purified from the soluble fraction of the cell homogenate by first heat-precipitating E. coli host proteins, followed by anion exchange and hydroxylapatite chromatography. The final preparation was more than 98% pure as judged by SDS-PAGE and gel filtration chromatography on Superdex 75 (data not shown). The presence of the N-terminal methionine residues of both purified tHisF and tHisH were confirmed by N-terminal protein sequencing. The complex of tHisF and tHisH (tHisH-tHisF) was prepared by mixing equal molar amounts of both proteins, followed by gel filtration chromatography in order to remove any unintentional surplus of either of the components. The concentration of the purified proteins was determined by absorption spectroscopy, using calculated molar extinction coefficients at 280 nm (26Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar) of 11,500m−1 cm−1for tHisF, 17,400 m−1cm−1 for tHisH, and 28,900m−1 cm−1for the tHisH-tHisF complex. The molecular masses and the association states of recombinant tHisF, tHisH, and tHisH-tHisF were determined by analytical gel filtration chromatography and equilibrium analytical ultracentrifugation. tHisF eluted from a calibrated Superdex 75 column with an apparent molecular mass of 26.4 kDa (27Höcker B. Beismann-Driemeyer S. Hettwer S. Lustig A. Sterner R. Nat. Struct. Biol. 2001; 8: 32-36Crossref PubMed Scopus (131) Google Scholar), which is similar to the molecular mass for the monomer as calculated from the amino acid sequence (27.7 kDa). tHisH eluted with a significantly lower apparent molecular mass (17.4 kDa) than the calculated molecular mass for the monomer (23.1 kDa). A similarly retarded elution has been observed previously for eHisH (11Klem T.J. Davisson V.J. Biochemistry. 1993; 32: 5177-5186Crossref PubMed Scopus (97) Google Scholar), for unknown reasons. Mixed equal molar amounts of tHisH and tHisF eluted as a single peak with an apparent molecular mass of 41.8 kDa, which is smaller than the calculated molecular mass for the 1:1 complex (50.8 kDa). In order to further clarify their association states, the molecular masses of tHisH, tHisF, and tHisH-tHisF were determined by analytical ultracentrif" @default.
• W2034144975 created "2016-06-24" @default.
• W2034144975 creator A5002529505 @default.
• W2034144975 creator A5009252615 @default.
• W2034144975 date "2001-01-01" @default.
• W2034144975 modified "2023-10-09" @default.
• W2034144975 title "Imidazole Glycerol Phosphate Synthase fromThermotoga maritima" @default.
• W2034144975 cites W1486946238 @default.
• W2034144975 cites W1953738731 @default.
• W2034144975 cites W1964205591 @default.
• W2034144975 cites W1971496061 @default.
• W2034144975 cites W1972580421 @default.
• W2034144975 cites W1974525344 @default.
• W2034144975 cites W1975085160 @default.
• W2034144975 cites W1976299470 @default.
• W2034144975 cites W1983687097 @default.
• W2034144975 cites W1987733787 @default.
• W2034144975 cites W1995298289 @default.
• W2034144975 cites W1995730663 @default.
• W2034144975 cites W2003668909 @default.
• W2034144975 cites W2019452958 @default.
• W2034144975 cites W2023002749 @default.
• W2034144975 cites W2024888615 @default.
• W2034144975 cites W2026554647 @default.
• W2034144975 cites W2026808634 @default.
• W2034144975 cites W2032854305 @default.
• W2034144975 cites W2050265985 @default.
• W2034144975 cites W2052919977 @default.
• W2034144975 cites W2053623997 @default.
• W2034144975 cites W2081604308 @default.
• W2034144975 cites W2084151820 @default.
• W2034144975 cites W2085818775 @default.
• W2034144975 cites W2088749908 @default.
• W2034144975 cites W2092642545 @default.
• W2034144975 cites W2093653062 @default.
• W2034144975 cites W2094633529 @default.
• W2034144975 cites W2099924664 @default.
• W2034144975 cites W2100837269 @default.
• W2034144975 cites W2104017463 @default.
• W2034144975 cites W2131367352 @default.
• W2034144975 cites W2146228523 @default.
• W2034144975 cites W2195878577 @default.
• W2034144975 cites W2290318361 @default.
• W2034144975 cites W2414307244 @default.
• W2034144975 cites W4293247451 @default.
• W2034144975 doi "https://doi.org/10.1074/jbc.m102012200" @default.
• W2034144975 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11264293" @default.
• W2034144975 hasPublicationYear "2001" @default.
• W2034144975 type Work @default.
• W2034144975 sameAs 2034144975 @default.
• W2034144975 citedByCount "87" @default.
• W2034144975 countsByYear W20341449752012 @default.
• W2034144975 countsByYear W20341449752013 @default.
• W2034144975 countsByYear W20341449752014 @default.
• W2034144975 countsByYear W20341449752015 @default.
• W2034144975 countsByYear W20341449752016 @default.
• W2034144975 countsByYear W20341449752017 @default.
• W2034144975 countsByYear W20341449752018 @default.
• W2034144975 countsByYear W20341449752019 @default.
• W2034144975 countsByYear W20341449752020 @default.
• W2034144975 countsByYear W20341449752021 @default.
• W2034144975 countsByYear W20341449752022 @default.
• W2034144975 countsByYear W20341449752023 @default.
• W2034144975 crossrefType "journal-article" @default.
• W2034144975 hasAuthorship W2034144975A5002529505 @default.
• W2034144975 hasAuthorship W2034144975A5009252615 @default.
• W2034144975 hasBestOaLocation W20341449751 @default.
• W2034144975 hasConcept C112243037 @default.
• W2034144975 hasConcept C181199279 @default.
• W2034144975 hasConcept C185592680 @default.
• W2034144975 hasConcept C2777132085 @default.
• W2034144975 hasConcept C2780874372 @default.
• W2034144975 hasConcept C2780881558 @default.
• W2034144975 hasConcept C55493867 @default.
• W2034144975 hasConceptScore W2034144975C112243037 @default.
• W2034144975 hasConceptScore W2034144975C181199279 @default.
• W2034144975 hasConceptScore W2034144975C185592680 @default.
• W2034144975 hasConceptScore W2034144975C2777132085 @default.
• W2034144975 hasConceptScore W2034144975C2780874372 @default.
• W2034144975 hasConceptScore W2034144975C2780881558 @default.
• W2034144975 hasConceptScore W2034144975C55493867 @default.
• W2034144975 hasIssue "23" @default.
• W2034144975 hasLocation W20341449751 @default.
• W2034144975 hasOpenAccess W2034144975 @default.
• W2034144975 hasPrimaryLocation W20341449751 @default.
• W2034144975 hasRelatedWork W1939047279 @default.
• W2034144975 hasRelatedWork W1967467262 @default.
• W2034144975 hasRelatedWork W2059520724 @default.
• W2034144975 hasRelatedWork W2067293160 @default.
• W2034144975 hasRelatedWork W2077083979 @default.
• W2034144975 hasRelatedWork W2386553017 @default.
• W2034144975 hasRelatedWork W3144613255 @default.
• W2034144975 hasRelatedWork W4251038669 @default.
• W2034144975 hasRelatedWork W4377724404 @default.
• W2034144975 hasRelatedWork W989046742 @default.
• W2034144975 hasVolume "276" @default.
• W2034144975 isParatext "false" @default.
• W2034144975 isRetracted "false" @default.

• next