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• W2171596444 abstract "Combinatorial mutagenesis and in vivoselection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg90. Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand ink cat. Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its ε-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed. Combinatorial mutagenesis and in vivoselection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg90. Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand ink cat. Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its ε-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed. B. subtilis chorismate mutase of the AroH class base pair(s) dithiothreitol wild type Protein Data Bank Chorismate mutase catalyzes one of few pericyclic reactions in biology. The transformation of chorismate 1 to prephenate3 (Fig. 1 A), formally a Claisen rearrangement, constitutes the first committed step in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and higher plants (1Haslam E. Shikimic Acid: Metabolism and Metabolites. John Wiley & Sons, Inc., New York1993Google Scholar). The reaction is strongly exergonic (2Kast P. Tewari Y.B. Wiest O. Hilvert D. Houk K.N. Goldberg R.N. J. Phys. Chem. B. 1997; 101: 10976-10982Crossref Scopus (57) Google Scholar) and proceeds in the absence of enzymatic catalysis as a concerted but asynchronous process (3Addadi L. Jaffe E.K. Knowles J.R. Biochemistry. 1983; 22: 4494-4501Crossref PubMed Scopus (110) Google Scholar, 4Gajewski J.J. Jurayj J. Kimbrough D.R. Gande M.E. Ganem B. Carpenter B.K. J. Am. Chem. Soc. 1987; 109: 1170-1186Crossref Scopus (204) Google Scholar) via a chair-like transition state (see figs. 1 and 2) (5Copley S.D. Knowles J.R. J. Am. Chem. Soc. 1985; 107: 5306-5308Crossref Scopus (80) Google Scholar).Figure 1A, rearrangement of (−)-chorismate (1) to prephenate (3) involving a chair-like transition state (2). B, transition state analog for the chorismate mutase reaction. The oxabicyclic dicarboxylic acid 4 is the tightest binding inhibitor known for BsCM (54Bartlett P.A. Johnson C.R. J. Am. Chem. Soc. 1985; 107: 7792-7793Crossref Scopus (112) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Natural chorismate mutases accelerate the rearrangement of chorismate by more than a million-fold (6Andrews P.R. Smith G.D. Young I.G. Biochemistry. 1973; 12: 3492-3498Crossref PubMed Scopus (201) Google Scholar). Determination of the crystal structures of three of these catalysts (7Chook Y.M. Ke H. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8600-8603Crossref PubMed Scopus (182) Google Scholar, 8Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (158) Google Scholar, 9Lee A.Y. Karplus P.A. Ganem B. Clardy J. J. Am. Chem. Soc. 1995; 117: 3627-3628Crossref Scopus (197) Google Scholar, 10Xue Y. Lipscomb W.N. Graf R. Schnappauf G. Braus G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10814-10818Crossref PubMed Scopus (66) Google Scholar, 11Sträter N. Schnappauf G. Braus G. Lipscomb W.N. Structure ( Lond. ). 1997; 5: 1437-1452Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) in conjunction with extensive comparison of primary structures derived from available genomic sequences from a variety of organisms (12Gu W. Williams D.S. Aldrich H.C. Xie G. Gabriel D.W. Jensen R.A. Microb. Comp. Genomics. 1997; 2: 141-158Crossref PubMed Scopus (24) Google Scholar, 13MacBeath G. Kast P. Hilvert D. Biochemistry. 1998; 37: 10062-10073Crossref PubMed Scopus (79) Google Scholar) has shown that the enzymes fall into two structurally distinct classes (13MacBeath G. Kast P. Hilvert D. Biochemistry. 1998; 37: 10062-10073Crossref PubMed Scopus (79) Google Scholar). The overwhelming majority of chorismate mutases (from all three domains of life) belong to the AroQ family, whose prototype is the all-helix-bundle chorismate mutase domain of the Escherichia coli chorismate mutase-prephenate dehydratase (9Lee A.Y. Karplus P.A. Ganem B. Clardy J. J. Am. Chem. Soc. 1995; 117: 3627-3628Crossref Scopus (197) Google Scholar). The AroH class, currently comprising only a monofunctional chorismate mutase fromBacillus subtilis(BsCM),1 putative coding regions from two other Bacillus species, Streptomyces coelicolor, and the cyanobacterium Synechocystis sp. strain PCC6803, has a trimeric pseudo α/β-barrel topology with three active sites at the subunit interfaces (7Chook Y.M. Ke H. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8600-8603Crossref PubMed Scopus (182) Google Scholar, 8Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (158) Google Scholar). Even though the mechanism of the mutase reaction has been extensively studied over several decades, the origin of the catalytic rate enhancement still remains a matter of debate (14Görisch H. Biochemistry. 1978; 17: 3700-3705Crossref PubMed Scopus (76) Google Scholar, 15Kast P. Asif-Ullah M. Hilvert D. Tetrahedron Lett. 1996; 37: 2691-2694Crossref Scopus (103) Google Scholar, 16Ganem B. Angew. Chem. Int. Ed. Engl. 1996; 35: 936-945Crossref Scopus (103) Google Scholar, 17Wiest O. Houk K.N. J. Am. Chem. Soc. 1995; 117: 11628-11639Crossref Scopus (76) Google Scholar, 18Lyne P.D. Mulholland A.J. Richards W.G. J. Am. Chem. Soc. 1995; 117: 11345-11350Crossref Scopus (147) Google Scholar, 19Khanjin N.A. Snyder J.P. Menger F.M. J. Am. Chem. Soc. 1999; 121: 11831-11846Crossref Scopus (72) Google Scholar). The mechanistic questions that remain unanswered include the extent to which conformational constraint of the flexible substrate (15Kast P. Asif-Ullah M. Hilvert D. Tetrahedron Lett. 1996; 37: 2691-2694Crossref Scopus (103) Google Scholar, 19Khanjin N.A. Snyder J.P. Menger F.M. J. Am. Chem. Soc. 1999; 121: 11831-11846Crossref Scopus (72) Google Scholar), specific hydrogen bonding to the transition state (20Lee A.Y. Stewart J.D. Clardy J. Ganem B. Chem. Biol. ( Lond. ). 1995; 2: 195-203Abstract Full Text PDF PubMed Scopus (110) Google Scholar), and electrostatic interactions (see below) contribute to catalysis. The well characterized BsCM enzyme constitutes an excellent system to study such questions. From viscosity-variation experiments, it is known that BsCM is only partially diffusion-controlled (21Mattei P. Kast P. Hilvert D. Eur. J. Biochem. 1999; 261: 25-32Crossref PubMed Scopus (54) Google Scholar). Heavy-atom isotope effect experiments have established that chemistry is significantly rate-determining in this case, with cleavage of the C-O bond preceding formation of the new C-C bond in the transition state (22Gustin D.J. Mattei P. Kast P. Wiest O. Lee L. Cleland W.W. Hilvert D. J. Am. Chem. Soc. 1999; 121: 1756-1757Crossref Scopus (83) Google Scholar) as deduced previously for the uncatalyzed rearrangement (3Addadi L. Jaffe E.K. Knowles J.R. Biochemistry. 1983; 22: 4494-4501Crossref PubMed Scopus (110) Google Scholar, 4Gajewski J.J. Jurayj J. Kimbrough D.R. Gande M.E. Ganem B. Carpenter B.K. J. Am. Chem. Soc. 1987; 109: 1170-1186Crossref Scopus (204) Google Scholar). Furthermore, the magnitude of the observed isotope effect suggests that the transition state of the enzyme-catalyzed process is more highly polarized than that of the uncatalyzed reaction (22Gustin D.J. Mattei P. Kast P. Wiest O. Lee L. Cleland W.W. Hilvert D. J. Am. Chem. Soc. 1999; 121: 1756-1757Crossref Scopus (83) Google Scholar). Electrostatic interactions may therefore be essential for efficient catalysis (8Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (158) Google Scholar,23Haynes M.R. Stura E.A. Hilvert D. Wilson I.A. Science. 1994; 263: 646-652Crossref PubMed Scopus (154) Google Scholar). Complementarily charged active site residues could conceivably stabilize developing charges in transition state 2 (Fig.1 A). Computational studies have highlighted stabilizing interactions between the positively charged Arg at position 90 of BsCM and the partially negatively charged ether oxygen of chorismate in the transition state (17Wiest O. Houk K.N. J. Am. Chem. Soc. 1995; 117: 11628-11639Crossref Scopus (76) Google Scholar, 18Lyne P.D. Mulholland A.J. Richards W.G. J. Am. Chem. Soc. 1995; 117: 11345-11350Crossref Scopus (147) Google Scholar, 24Davidson M.M. Gould I.R. Hillier I.H. J. Chem. Soc. Perkin Trans. I. 1996; 2: 525-531Crossref Scopus (30) Google Scholar). In previous work, we addressed the hypothesis of electrostatic catalysis by combinatorial mutagenesis experiments (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar, 26Kast P. Hartgerink J.D. Asif-Ullah M. Hilvert D. J. Am. Chem. Soc. 1996; 118: 3069-3070Crossref Scopus (42) Google Scholar). An evolutionary approach was employed consisting of randomizing critical positions in the active site of BsCM combined with direct genetic selection for mutant genes encoding functional catalysts. From the spectrum of active BsCM mutants obtained it was obvious that survival of host cells under selective conditions was only possible for variants with a positively charged group in the region of the ether oxygen of bound chorismate (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). Here, we report detailed kinetic and structural characterization of several relevant mutant enzymes derived from our combinatorial libraries. The results suggest that a single cation provides the dominant catalytic interaction in the active site of chorismate mutases. Chorismic acid was prepared according to published procedures (27Grisostomi C. Kast P. Pulido R. Huynh J. Hilvert D. Bioorg. Chem. 1997; 25: 297-305Crossref Scopus (42) Google Scholar). The transition state analog for the chorismate mutase reaction, the oxabicyclic dicarboxylic acid4 of Fig. 1 B, was synthesized as described previously (28Smith W.W. Bartlett P.A. J. Org. Chem. 1993; 58: 7308-7309Crossref Scopus (14) Google Scholar). Oligonucleotides were custom-synthesized by Life Technologies, Inc. Restriction endonucleases and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA), and PfuDNA polymerase, purchased from Stratagene (La Jolla, CA), was used for polymerase chain reaction. E. coli strain KA13 (13MacBeath G. Kast P. Hilvert D. Biochemistry. 1998; 37: 10062-10073Crossref PubMed Scopus (79) Google Scholar,29MacBeath G. Kast P. Biotechniques. 1998; 24: 789-794Crossref PubMed Scopus (45) Google Scholar) was used for the construction of the aroH expression plasmids as well as for BsCM protein production. The in vivocomplementation system KA12/pKIMP-UAUC was described previously (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). Both KA13 and KA12 have chromosomal deletions of the endogenousE. coli chorismate mutase genes. The cloning vector pET-22b(+) was from Novagen (Madison, WI). Plasmids pKCMT-W, pKCMT-R90A, pKCMT-R90G, pKCMT-C88S/R90K, and pKCMT-C88K/R90S carrytrc promoter-expressed B. subtilis aroH gene variants encoding wild-type (WT), R90A, R90G, C88S/R90K, and C88K/R90S BsCM, respectively. pKCMT-W and the selected mutant pKCMT plasmids from two different combinatorial libraries were characterized previously (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). pKET3-W (21Mattei P. Kast P. Hilvert D. Eur. J. Biochem. 1999; 261: 25-32Crossref PubMed Scopus (54) Google Scholar) is a pET-22b(+) derivative that contains the WTaroH gene under the control of the T7 RNA polymerase promoter. Individual mutantaroH genes were tested for their ability to complement the chorismate mutase deficiency of strain KA12 after transforming KA12/pKIMP-UAUC with the relevant pKCMT plasmids. The assay procedure involved comparing growth of single colonies on minimal medium (M9c) agar plates in the presence or absence of 20 μg/ml l-Phe and l-Tyr. M9c plates were composed of 1 × M9 salts (6 mg/ml Na2HPO4, 3 mg/ml KH2PO4, 1 mg/ml NH4Cl, 0.5 mg/ml NaCl, pH 7.0 (30Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar)) supplemented with 0.2% (w/v) d-(+)-Glc, 1 mm MgSO4, 0.1 mmCaCl2, 5 μg/ml thiamine-HCl, 5 μg/ml 4-hydroxybenzoic acid, 5 μg/ml 4-aminobenzoic acid, 1.6 μg/ml 2,3-dihydroxybenzoic acid, 20 μg/mll-Trp, 100 μg/ml sodium ampicillin, 20 μg/ml chloramphenicol, and 15 g of agar/liter. Media and assay conditions corresponded to those used previously (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). For high level production of mutant BsCM proteins, the corresponding aroH genes were transferred from the initialtrc promoter expression plasmids (pKCMT types) to pET-22b(+), allowing for T7 promoter-driven gene expression. ThearoH genes for C88S/R90K or C88K/R90S variants were amplified by polymerase chain reaction with oligonucleotides T7-aroHW (5′-GGAAACAGCATATGATGATTCGCGGAATT) and TMUTN (5′-ATCAAGCGGCCGCACTAGT) using HincII-digested pKCMT-C88S/R90K or pKCMT-C88K/R90S as templates, respectively. The 414-bp polymerase chain reaction products were digested withNdeI and EagI to yield 394-bp fragments that were then ligated to the 5372-bp NdeI-EagI fragment of pET-22b(+), resulting in plasmids pKET3-C88S/R90K and pKET3-C88K/R90S. pKET3-R90A and pKET3-R90G were obtained by replacing the 343-bpEcoRI-HindIII restriction fragment in pKET3-W with the corresponding fragments from pKCMT-R90A and pKCMT-R90G, respectively. We have observed that a single silent base change in the codon for Ile3 (from ATC to ATT) caused a 3-fold increase in WT protein yield (data not shown), even though codon usage in highly expressed genes in E. coli favors ATC (83%) over ATT (17%) (according to Version 8 of the Wisconsin Sequence Analysis Package by Genetics Computer Group, Inc, Madison, WI). Consequently, the third codon in aroH on the pKCMT plasmids (ATC) was mutated to ATT in all pKET3 constructs. The entire aroH gene and relevant gene expression signals in the new pKET3 plasmids (5766 bp) were confirmed by sequencing both strands with primers T7PRO2 (5′-TAATACGACTCACTATAGGG) and T7TER (5′-CAGCAGCCAACTCAGCT) using an ABI 373 DNA sequencer (PerkinElmer Life Sciences) with dye terminator nucleotides and chain termination chemistry (31Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52769) Google Scholar). All DNA manipulations were carried out according to standard procedures (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 5 ml of LB medium (30Miller J.H. A Short Course in Bacterial Genetics: A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992Google Scholar) containing 150 μg/ml sodium ampicillin was inoculated with a single colony of E. coli strain KA13 transformed with the relevant pKET3 plasmid and incubated overnight at 37 °C. 1 liter of the same medium with 150 μg/ml sodium ampicillin was seeded with the densely grown preculture and shaken at 250 rpm at 37 °C until anA 600 nm of 0.6 was reached. Then, isopropyl-β-d-thiogalactopyranoside was added (500 μm final concentration) to induce synthesis of the chromosomally encoded T7 RNA polymerase in KA13 for expression of the T7 promoter-controlled aroH genes. The incubation was continued for another 24 h. The cells were harvested by centrifugation (20 min, 2,500 × g, 4 °C), and the cell pellet was stored overnight at −20 °C. The thawed cells were gently resuspended in 180 ml of osmobuffer (20% (w/v) sucrose, 30 mm Tris-HCl, pH 8.0) at 0 °C. EDTA and dithiothreitol (DTT) were added to 1 mm final concentration each. This cell suspension was shaken at room temperature for 15 min at 200 rpm followed by centrifugation (10 min, 8,000 × g, 4 °C). The supernatant containing the soluble protein fraction was transferred to a new centrifuge bottle and spun again to remove remaining traces of cells and debris. The clear supernatant was subsequently concentrated to 20 ml by ultrafiltration at 4 °C (200 ml ultrafiltration unit using a Diaflo YM10 membrane; Amicon, Beverly, MA). The concentrate was dialyzed twice (at 4 °C; Spectra/Por 1, cutoff 6–8 kDa; Spectrum Laboratories, Inc., Laguna Hills, CA) for at least 4 h against 1 liter of degassed buffer B (50 mmglycine-NaOH, pH 8.9, 1 mm DTT, 5% (v/v) 2-propanol, 10% (v/v) glycerol). The sample was then filtered through 0.8/0.2-μm Acrodisc PF syringe filters (Gelman Sciences, Ann Arbor, MI) to remove insoluble precipitates. The filtrate was loaded onto a diethylaminoethyl (DEAE) DE-52 cellulose anion exchange column (2.5-ml bed volume; catalog number 4057–050, Whatman Inc., Clifton, NJ) previously equilibrated in degassed buffer B. The gravity flow column was washed extensively with buffer B and then eluted with an NaCl gradient (0–300 mm) in buffer B. Fractions containing pure BsCM were combined and concentrated by ultrafiltration at 4 °C using Centriprep-10 devices (Amicon). BsCM concentration was determined by UV absorption using a calculated molar extinction coefficient (per subunit: 8250 m−1cm−1). For storage, phenylmethanesulfonyl fluoride, DTT, and NaN3 were added to give final concentrations of 0.6 mm, 10 mm and 0.02%, respectively. Purification was monitored by analyzing appropriate samples and fractions on 20% SDS polyacrylamide gels (PhastSystem, Amersham Pharmacia Biotech). Proteins were prepared for electrospray ionization mass spectrometry by desalting on a NAP-5 column (Amersham Pharmacia Biotech) that was equilibrated and eluted with 5% aqueous acetic acid. Electrospray ionization mass spectrometry was performed by The Scripps Mass Spectrometry Laboratory on an API 100 PerkinElmer Life Sciences SCIEX single quadrupole mass spectrometer. Experimental results (which had an accuracy of 0.01%) were compared with theoretical protein masses calculated with the program PeptideSort (Genetics Computer Group, Inc.). Circular dichroism spectra were recorded on an Aviv circular dichroism spectropolarimeter model 61DS equipped with a thermoelectric cuvette holder. BsCM samples were measured at a protein concentration of 6.7 μm (trimer) at 23 °C in phosphate-buffered saline (10 mm phosphate, 160 mm NaCl, pH 7.5) in a cuvette with a path length of 0.1 cm. Spectra were obtained by averaging 5 wavelength scans from 260 nm to 200 nm in 0.5-nm steps, with a signal averaging time of 2 s and a bandwidth of 1.5 nm. Protein for crystallization was dialyzed against a solution containing 10 mm Tris-HCl, pH 7.5, 2 mm DTT, and 0.125 mm EDTA. Crystallization experiments were performed at room temperature using the hanging drop vapor diffusion technique. The hanging drop was formed by mixing 3 μl of the protein solution (at 15–20 mg/ml) with 3 μl of the reservoir solution consisting of 30% polyethylene glycol 400, 50 mm Tris-HCl, pH 7.0 to pH 7.5, and 50 mmMgCl2. Prism-shaped crystals with a typical size of 0.2 × 0.4 × 0.5 mm appeared after 3–6 days. The space group was determined to be R3, with cell parameters ofa = b = 82.6 Å, and c= 42.8 Å (one molecule per asymmetric unit). X-ray data extending to 1.8-Å resolution were recorded on an Raxis-IIc image plate detector mounted on a Rigaku rotating anode generator (RU200) operating at 50 kV/100 mA. Data were collected at room temperature with the crystal-to-detector distance set to 90 mm and an oscillation angle of 2°. All data were processed and scaled using DENZO and SCALEPACK (33Otwinowski Z. DENZO: An Oscillation Data Processing Suite for Macromolecular Crystallography , Version 1.3.0. Yale University, New Haven, CT1993Google Scholar, 34Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The final data sets include 10,748 and 9,357 unique reflections, respectively, for the mutants C88S/R90K and C88K/R90S. This corresponds to a completeness of 97.7 and 100.0% to 1.75- and 1.85-Å resolution (redundancies are 4.9 and 4.2), respectively. The respective symmetry R-factors are 6.0 and 8.5% for the two data sets. The structure of monomer A of the published wild-type protein (Protein Data Bank (PDB) code 2CHT (7Chook Y.M. Ke H. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8600-8603Crossref PubMed Scopus (182) Google Scholar, 8Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (158) Google Scholar)) was used as the search model for molecular replacement. Molecular replacement was performed using X-PLOR (35Brünger A.T. X-PLOR: A System for Crystallography and NMR , Version 3.1. Yale University, New Haven, CT1993Google Scholar) and AMoRe (36Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) in separate runs. Both programs yielded virtually the same solutions, which were verified by inspecting the resulting electron density maps. The structures were subsequently refined with X-PLOR (35Brünger A.T. X-PLOR: A System for Crystallography and NMR , Version 3.1. Yale University, New Haven, CT1993Google Scholar, 37Brünger A.T. Kuriyan J. Karplus M. Science. 1987; 235: 458-460Crossref PubMed Scopus (2126) Google Scholar) using torsion-angle dynamics (38Rice L.M. Brünger A.T. Proteins. 1994; 19: 277-290Crossref PubMed Scopus (382) Google Scholar). For the final rounds of refinement, data were restricted to 1.9 and 2.0 Å for the mutants C88S/R90K and C88K/R90S, respectively, because beyond that resolution, both R and R free increased dramatically, coinciding with a decrease in data quality as judged fromR sym. Model building was carried out on a molecular graphics workstation using O (39Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Superpositions of crystal structure coordinates of WT BsCM and mutant variants were performed with the program O (39Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). In all cases, 2664 peptide backbone atoms of segments encompassing residues Met2 to Ala112 of each subunit in the trimer were superimposed. For the comparisons, we selected the trimer composed of subunits J, K, and L from the asymmetric units of the WT structure complexed with 4 (7Chook Y.M. Ke H. Lipscomb W.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8600-8603Crossref PubMed Scopus (182) Google Scholar, 8Chook Y.M. Gray J.V. Ke H. Lipscomb W.N. J. Mol. Biol. 1994; 240: 476-500Crossref PubMed Scopus (158) Google Scholar) and crystallographic trimers of the BsCM mutants. 2The atomic coordinates for WT BsCM in a complex with inhibitor 4 (PDB code 2CHT) were from the Protein Data Bank. The coordinates for unliganded C88S/R90K and C88K/R90S can be accessed under PDB codes 1FNJ and 1FNK, respectively. Kinetic assays were carried out at 30 °C in 50 mm potassium phosphate, pH 7.5, by monitoring the disappearance of the substrate chorismate at 274 nm (ε274 nm = 2630 m−1cm−1) or 310 nm (ε310 nm = 370m−1 cm−1) by UV spectroscopy. All curve fittings of initial velocity data were done with the program KaleidaGraph (version 3.08; Synergy Software, Reading, PA). For determination of Michaelis-Menten parametersk cat (per active site) andK m, substrate concentrations up to 5.7 mm were used. K i for competitive inhibition of WT BsCM by transition state analog 4 was determined by fitting initial velocities obtained at 36 μm chorismate and nine different concentrations of inhibitor (0 to 100 μm) to the equation v= (k cat[E0][S])/([S] +K m(1 + [I]/K i)) (40Fersht A. Enzyme Structure and Mechanism. 2nd Ed. W. H. Freeman and Co., New York, NY1985: 107-109Google Scholar). For inhibition studies with C88K/R90S and C88S/R90K, concentrations up to 1 mm of 4 were used at a single chorismate concentration of 75 μm. The K i value for R90G was estimated from a replot of the slopes of a Dixon plot obtained by varying inhibitor concentrations (0 to 30 μm) at four fixed chorismate concentrations between 48 and 126 μm. Previous in vivo studies of mutant BsCM genes indicated that a positively charged amino acid side chain at position 90 or 88 is a prerequisite for detectable chorismate mutase activity (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). To assess and quantify the role of this cation for the enzymatic mechanism, we have examined several BsCM variants in greater detail. The corresponding aroH genes were taken from our collection of mutants generated for the combinatorial mutagenesis and selection experiments (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar), and their phenotype was redetermined (TableI).Table IFeatures of BsCM variants relevant to this studyBsCM catalystResidue at position 88Residue at position 90Phenotype in vivo 1-aThe growth at 30 °C ofE. coli KA12/pKIMP-UAUC transformants hostingaroH genes encoding the respective mutants was scored on an arbitrary, comparative scale ranging from excellent (++++), good (+++), moderate (++), weak (+), very weak (+/−) to no (−) growth. The reported data coincide with those collected previously using identical assay conditions (25).WTCysArg++++R90ACysAla−R90GCysGly−R90KCysLys−C88S/R90KSerLys++C88K/R90SLysSer+++Listed are the amino acids at the randomized positions 88 and 90 of relevant BsCM variants. The in vivo phenotype refers to growth (colony size) on minimal agar plates of the bacterial host system transformed with the respective aroH gene variants. The genetic complementation system was described previously (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar).1-a The growth at 30 °C ofE. coli KA12/pKIMP-UAUC transformants hostingaroH genes encoding the respective mutants was scored on an arbitrary, comparative scale ranging from excellent (++++), good (+++), moderate (++), weak (+), very weak (+/−) to no (−) growth. The reported data coincide with those collected previously using identical assay conditions (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). Open table in a new tab Listed are the amino acids at the randomized positions 88 and 90 of relevant BsCM variants. The in vivo phenotype refers to growth (colony size) on minimal agar plates of the bacterial host system transformed with the respective aroH gene variants. The genetic complementation system was described previously (25Kast P. Asif-Ullah M. Jiang N. Hilvert D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5043-5048Crossref PubMed Scopus (122) Google Scholar). The subset of enzymes chosen here includes two variants in which the positively charged guanidinium group at position 90 is absent (R90A and R90G). Both of these proteins are completely inactive in vivo (Table I). All active aroH genes isolated from libraries in which positions 88 or 90 were randomiz" @default.
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• W2171596444 date "2000-11-01" @default.
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• W2171596444 title "A Strategically Positioned Cation Is Crucial for Efficient Catalysis by Chorismate Mutase" @default.
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