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• W3092443721 abstract "Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity. Chorismate mutase (CM), an essential enzyme at the branch-point of the shikimate pathway, is required for the biosynthesis of phenylalanine and tyrosine in bacteria, archaea, plants, and fungi. MtCM, the CM from Mycobacterium tuberculosis, has less than 1% of the catalytic efficiency of a typical natural CM and requires complex formation with 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase for high activity. To explore the full potential of MtCM for catalyzing its native reaction, we applied diverse iterative cycles of mutagenesis and selection, thereby raising kcat/Km 270-fold to 5 × 105m−1s−1, which is even higher than for the complex. Moreover, the evolutionarily optimized autonomous MtCM, which had 11 of its 90 amino acids exchanged, was stabilized compared with its progenitor, as indicated by a 9 °C increase in melting temperature. The 1.5 Å crystal structure of the top-evolved MtCM variant reveals the molecular underpinnings of this activity boost. Some acquired residues (e.g. Pro52 and Asp55) are conserved in naturally efficient CMs, but most of them lie beyond the active site. Our evolutionary trajectories reached a plateau at the level of the best natural enzymes, suggesting that we have exhausted the potential of MtCM. Taken together, these findings show that the scaffold of MtCM, which naturally evolved for mediocrity to enable inter-enzyme allosteric regulation of the shikimate pathway, is inherently capable of high activity. The potential of a microbial cell to respond to changing environmental conditions is reflected in its ability to reorganize the metabolic flux. Allosteric feedback regulation of enzymes, where effector binding to an allosteric site tunes the activity of a distant active site, instantaneously achieves such a response. Typically, this type of regulation employs allosteric sites on the enzyme itself. Alternatively, an allosteric site can be temporarily provided by transient protein-protein interactions, also called “inter-enzyme allostery”, as first described for shikimate pathway enzymes from Mycobacterium tuberculosis H37Rv (1Munack S. Roderer K. Ökvist M. Kamarauskaite˙ J. Sasso S. van Eerde A. Kast P. Krengel U. Remote control by inter-enzyme allostery: a novel paradigm for regulation of the shikimate pathway.J. Mol. Biol. 2016; 428 (26776476): 1237-125510.1016/j.jmb.2016.01.001Crossref PubMed Scopus (17) Google Scholar) and more recently also from Corynebacterium glutamicum (2Burschowsky D. Thorbjørnsrud H.V. Heim J.B. Fahrig-Kamarauskaitė J. Würth-Roderer K. Kast P. Krengel U. Inter-enzyme allosteric regulation of chorismate mutase in Corynebacterium glutamicum: structural basis of feedback activation by Trp.Biochemistry. 2018; 57 (29178787): 557-57310.1021/acs.biochem.7b01018Crossref PubMed Scopus (8) Google Scholar). Indeed, tight feedback regulation of metabolic flux is particularly important for the shikimate pathway, because it metabolizes a significant portion of organic carbon for the biosynthesis of energetically costly aromatic compounds in bacteria, archaea, fungi, algae, plants, and protozoan parasites (3Haslam E. Shikimic Acid: Metabolism and Metabolites. John Wiley, New York1993Google Scholar, 4Herrmann K.M. Weaver L.M. The shikimate pathway.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50 (15012217): 473-50310.1146/annurev.arplant.50.1.473Crossref PubMed Scopus (802) Google Scholar, 5Roberts F. Roberts C.W. Johnson J.J. Kyle D.E. Krell T. Coggins J.R. Coombs G.H. Milhous W.K. Tzipori S. Ferguson D.J. Chakrabarti D. McLeod R. Evidence for the shikimate pathway in apicomplexan parasites.Nature. 1998; 393 (9655396): 801-80510.1038/31723Crossref PubMed Scopus (161) Google Scholar). The pathway starts with the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) catalyzed by DAHP synthase (DS). Six subsequent enzymatic steps lead to the biosynthesis of the branch-point metabolite chorismate, the last common precursor of the aromatic amino acids and other essential aromatic compounds. The first committed step toward l-phenylalanine and l-tyrosine is the conversion of chorismate to prephenate (Fig. 1A), catalyzed by chorismate mutase (CM). Because DS and CM are the central nodes of the shikimate pathway, organisms have developed a variety of strategies both at the genetic (4Herrmann K.M. Weaver L.M. The shikimate pathway.Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50 (15012217): 473-50310.1146/annurev.arplant.50.1.473Crossref PubMed Scopus (802) Google Scholar) and protein (6Light S.H. Anderson W.F. The diversity of allosteric controls at the gateway to aromatic amino acid biosynthesis.Protein Sci. 2013; 22 (23400945): 395-40410.1002/pro.2233Crossref PubMed Scopus (32) Google Scholar, 7Fan Y. Cross P.J. Jameson G.B. Parker E.J. Exploring modular allostery via interchangeable regulatory domains.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (29507215): 3006-301110.1073/pnas.1717621115Crossref PubMed Scopus (14) Google Scholar, 8Sterritt O.W. Kessans S.A. Jameson G.B. Parker E.J. A pseudoisostructural type II DAH7PS enzyme from Pseudomonas aeruginosa: alternative evolutionary strategies to control shikimate pathway flux.Biochemistry. 2018; 57 (29608284): 2667-267810.1021/acs.biochem.8b00082Crossref PubMed Scopus (8) Google Scholar, 9Nazmi A.R. Lang E.J.M. Bai Y. Allison T.M. Othman M.H. Panjikar S. Arcus V.L. Parker E.J. Interdomain conformational changes provide allosteric regulation en route to chorismate.J. Biol. Chem. 2016; 291 (27502275): 21836-2184710.1074/jbc.M116.741637Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 10Bai Y. Lang E.J.M. Nazmi A.R. Parker E.J. Domain cross-talk within a bifunctional enzyme provides catalytic and allosteric functionality in the biosynthesis of aromatic amino acids.J. Biol. Chem. 2019; 294 (30670586): 4828-484210.1074/jbc.RA118.005220Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar) level to regulate these enzymes. In M. tuberculosis, CM (MtCM, a dimer encoded by Rv0948c, aroQδ) has evolved to transiently interact with DS (MtDS, a tetramer encoded by Rv2178c, aroG) to form a heterooctameric complex (Fig. 1B). Only the complexed, but not the free dimeric MtCM, is responsive to feedback regulation by Phe and Tyr (1Munack S. Roderer K. Ökvist M. Kamarauskaite˙ J. Sasso S. van Eerde A. Kast P. Krengel U. Remote control by inter-enzyme allostery: a novel paradigm for regulation of the shikimate pathway.J. Mol. Biol. 2016; 428 (26776476): 1237-125510.1016/j.jmb.2016.01.001Crossref PubMed Scopus (17) Google Scholar, 11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar). MtCM represents the structurally simple AroQδ subclass of CMs composed of two intertwined three-helix subunits (Fig. 1C) (1Munack S. Roderer K. Ökvist M. Kamarauskaite˙ J. Sasso S. van Eerde A. Kast P. Krengel U. Remote control by inter-enzyme allostery: a novel paradigm for regulation of the shikimate pathway.J. Mol. Biol. 2016; 428 (26776476): 1237-125510.1016/j.jmb.2016.01.001Crossref PubMed Scopus (17) Google Scholar, 11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar). Other prototypical fold variants within the structurally and evolutionarily related α-helical AroQ class of CMs (12Roderer K. Kast P. Evolutionary cycles for pericyclic reactions - Or why we keep mutating mutases.Chimia. 2009; 63: 313-31710.2533/chimia.2009.313Crossref Scopus (4) Google Scholar) comprise the CM domain of the bifunctional CM-prephenate dehydratase from Escherichia coli (EcCM, subclass AroQα (13Lee A.Y. Karplus P.A. Ganem B. Clardy J. Atomic structure of the buried catalytic pocket of Escherichia coli chorismate mutase.J. Am. Chem. Soc. 1995; 117: 3627-362810.1021/ja00117a038Crossref Scopus (192) Google Scholar)), the elaborate eukaryotic 12-helical CM from Saccharomyces cerevisiae (ScCM, AroQβ (14Sträter N. Schnappauf G. Braus G. Lipscomb W.N. Mechanisms of catalysis and allosteric regulation of yeast chorismate mutase from crystal structures.Structure. 1997; 5 (9384560): 1437-145210.1016/S0969-2126(97)00294-3Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15Xue Y. Lipscomb W.N. Graf R. Schnappauf G. Braus G. The crystal structure of allosteric chorismate mutase at 2.2 Å resolution.Proc. Natl. Acad. Sci. U. S. A. 1994; 91 (7971967): 10814-1081810.1073/pnas.91.23.10814Crossref PubMed Scopus (64) Google Scholar)), and the secreted enzyme from M. tuberculosis (*MtCM, AroQγ (16Sasso S. Ramakrishnan C. Gamper M. Hilvert D. Kast P. Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.FEBS J. 2005; 272 (15654876): 375-38910.1111/j.1742-4658.2004.04478.xCrossref PubMed Scopus (56) Google Scholar)). In stark contrast to the α, β, and γ CM subclasses, MtCM utilizes an arginine residue (Arg46) instead of an otherwise absolutely conserved lysine to promote the electrostatic catalysis (17Warshel A. Sharma P.K. Kato M. Xiang Y. Liu H. Olsson M.H.M. Electrostatic basis for enzyme catalysis.Chem. Rev. 2006; 106 (16895325): 3210-323510.1021/cr0503106Crossref PubMed Scopus (834) Google Scholar) of the Claisen rearrangement of chorismate (Fig. 1, D and E) (11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar). Furthermore, MtCM is shorter at the C terminus and lacks the active site residue homologous to Gln88 in EcCM (18Liu D.R. Cload S.T. Pastor R.M. Schultz P.G. Analysis of active site residues in Escherichia coli chorismate mutase by site-directed mutagenesis.J. Am. Chem. Soc. 1996; 118: 1789-179010.1021/ja953151oCrossref Scopus (54) Google Scholar, 19Zhang S. Kongsaeree P. Clardy J. Wilson D.B. Ganem B. Site-directed mutagenesis of monofunctional chorismate mutase engineered from the E. coli P-protein.Bioorg. Med. Chem. 1996; 4 (8831972): 1015-102010.1016/0968-0896(96)00099-5Crossref PubMed Scopus (31) Google Scholar), Glu109 in *MtCM (20Ökvist M. Dey R. Sasso S. Grahn E. Kast P. Krengel U. 1.6 Å crystal structure of the secreted chorismate mutase from Mycobacterium tuberculosis: novel fold topology revealed.J. Mol. Biol. 2006; 357 (16499927): 1483-149910.1016/j.jmb.2006.01.069Crossref PubMed Scopus (41) Google Scholar), and Glu246 in ScCM (21Schnappauf G. Sträter N. Lipscomb W.N. Braus G.H. A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions.Proc. Natl. Acad. Sci. U. S. A. 1997; 94 (9238004): 8491-849610.1073/pnas.94.16.8491Crossref PubMed Scopus (35) Google Scholar). Despite these dramatic deviations from the consensus active site, MtCM is capable of catalyzing the conversion of chorismate to prephenate with a high catalytic efficiency (kcat/Km = 2.4 × 105m−1 s−1) (11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar). However, this can only be achieved in the presence of MtDS. On its own, MtCM is a mediocre enzyme catalyzing the reaction by two orders of magnitude less efficiently (kcat/Km of 1.8 × 103m−1 s−1) than the prototypical CMs of the other subclasses (16Sasso S. Ramakrishnan C. Gamper M. Hilvert D. Kast P. Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.FEBS J. 2005; 272 (15654876): 375-38910.1111/j.1742-4658.2004.04478.xCrossref PubMed Scopus (56) Google Scholar, 18Liu D.R. Cload S.T. Pastor R.M. Schultz P.G. Analysis of active site residues in Escherichia coli chorismate mutase by site-directed mutagenesis.J. Am. Chem. Soc. 1996; 118: 1789-179010.1021/ja953151oCrossref Scopus (54) Google Scholar, 21Schnappauf G. Sträter N. Lipscomb W.N. Braus G.H. A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions.Proc. Natl. Acad. Sci. U. S. A. 1997; 94 (9238004): 8491-849610.1073/pnas.94.16.8491Crossref PubMed Scopus (35) Google Scholar). In fact, the poor activity of the MtCM dimer is essential for effective shikimate pathway regulation, exerted through inter-enzyme allostery in M. tuberculosis. We (1Munack S. Roderer K. Ökvist M. Kamarauskaite˙ J. Sasso S. van Eerde A. Kast P. Krengel U. Remote control by inter-enzyme allostery: a novel paradigm for regulation of the shikimate pathway.J. Mol. Biol. 2016; 428 (26776476): 1237-125510.1016/j.jmb.2016.01.001Crossref PubMed Scopus (17) Google Scholar, 11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar) and others (22Blackmore N.J. Nazmi A.R. Hutton R.D. Webby M.N. Baker E.N. Jameson G.B. Parker E.J. Complex formation between two biosynthetic enzymes modifies the allosteric regulatory properties of both: an example of molecular symbiosis.J. Biol. Chem. 2015; 290 (26032422): 18187-1819810.1074/jbc.M115.638700Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 23Jiao W. Blackmore N.J. Nazmi A.R. Parker E.J. Quaternary structure is an essential component that contributes to the sophisticated allosteric regulation mechanism in a key enzyme from Mycobacterium tuberculosis.PLoS ONE. 2017; 12 (28665948)e018005210.1371/journal.pone.0180052Crossref PubMed Scopus (10) Google Scholar) have shown that binding of the allosteric feedback inhibitors Phe and Tyr to MtDS induces MtCM-MtDS complex dissociation and thereby a shift from high to low intracellular CM activity, providing tight control over cytoplasmic aromatic amino acid concentrations. In this study, we probed the structural and mechanistic requirements for the activity switch of MtCM and whether or not interaction with MtDS is mandatory for efficient catalysis. We have employed diverse cycles of directed evolution to improve the mediocre efficiency of MtCM as the prototype for catalytically impaired AroQδ enzymes (1Munack S. Roderer K. Ökvist M. Kamarauskaite˙ J. Sasso S. van Eerde A. Kast P. Krengel U. Remote control by inter-enzyme allostery: a novel paradigm for regulation of the shikimate pathway.J. Mol. Biol. 2016; 428 (26776476): 1237-125510.1016/j.jmb.2016.01.001Crossref PubMed Scopus (17) Google Scholar, 2Burschowsky D. Thorbjørnsrud H.V. Heim J.B. Fahrig-Kamarauskaitė J. Würth-Roderer K. Kast P. Krengel U. Inter-enzyme allosteric regulation of chorismate mutase in Corynebacterium glutamicum: structural basis of feedback activation by Trp.Biochemistry. 2018; 57 (29178787): 557-57310.1021/acs.biochem.7b01018Crossref PubMed Scopus (8) Google Scholar, 11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar). Our results reveal mutation patterns and structural changes responsible for high activity and demonstrate that MtCM is inherently capable of efficient catalysis despite the lack of crucial active site residues, which are otherwise strongly conserved in the AroQ family. Thus, retaining such catalytic residues is not a strict requirement for achieving maximum catalytic prowess. More important is the proper positioning and orientation of analogous functional groups. To explore the intrinsic potential of MtCM for efficient catalysis, we enlisted the strategy of directed evolution, a powerful experimental tool for probing and improving key features of enzymes (24Kast P. Hilvert D. Genetic selection strategies for generating and characterizing catalysts.Pure Appl. Chem. 1996; 68: 2017-202410.1351/pac199668112017Crossref Scopus (13) Google Scholar, 25Taylor S.V. Kast P. Hilvert D. Investigating and engineering enzymes by genetic selection.Angew. Chem. Int. Ed. 2001; 40: 3310-333510.1002/1521-3773(20010917)40:18<3310::AID-ANIE3310>3.0.CO;2-PCrossref PubMed Scopus (148) Google Scholar, 26Moore J.C. Arnold F.H. Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents.Nat. Biotechnol. 1996; 14: 458-46710.1038/nbt0496-458Crossref PubMed Scopus (347) Google Scholar, 27Jäckel C. Kast P. Hilvert D. Protein design by directed evolution.Annu. Rev. Biophys. 2008; 37 (18573077): 153-17310.1146/annurev.biophys.37.032807.125832Crossref PubMed Scopus (282) Google Scholar, 28Renata H. Wang Z.J. Arnold F.H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution.Angew. Chem. Int. Ed. Engl. 2015; 54 (25649694): 3351-336710.1002/anie.201409470Crossref PubMed Scopus (291) Google Scholar, 29Bershtein S. Tawfik D.S. Advances in laboratory evolution of enzymes.Curr. Opin. Chem. Biol. 2008; 12 (18284924): 151-15810.1016/j.cbpa.2008.01.027Crossref PubMed Scopus (198) Google Scholar). We applied several cycles of mutagenesis and selection to identify mutations in MtCM that increase CM efficiency in the absence of interacting MtDS (Figure 2, Figure 3). Because CMs are essential metabolic enzymes, our evolutionary approach could take advantage of direct selection using the previously described E. coli CM knockout strain KA12/pKIMP-UAUC (30Kast P. Asif-Ullah M. Jiang N. Hilvert D. Exploring the active site of chorismate mutase by combinatorial mutagenesis and selection: the importance of electrostatic catalysis.Proc. Natl. Acad. Sci. U. S. A. 1996; 93 (8643526): 5043-504810.1073/pnas.93.10.5043Crossref PubMed Scopus (115) Google Scholar) (Fig. 2A). Transformation with plasmid libraries carrying mutagenized M. tuberculosis aroQδ genes allows for rapid identification of survivors and thereby of active enzymes from typically 106 variants per evolutionary round under the appropriate selective conditions (Fig. 2B). As the starting point for the directed evolution we constructed H6-MtCM, an N-terminally His-tagged version of the WT MtCM that facilitates biochemical characterization. The catalytic efficiency (kcat/Km = 9 × 102m−1 s−1) of H6-MtCM is preserved within a factor of two of the parental enzyme (Table 1). H6-MtCM does not support the growth of KA12/pKIMP-UAUC on minimal medium lacking Tyr and Phe when its gene is introduced on the high-copy plasmid pKTNTET, downstream of the tightly repressed Ptet promoter (Fig. 2). pKTNTET additionally carries the genes tetR encoding the tet repressor and bla for ampicillin resistance. For convenient subsequent MtCM overproduction, pKTNTET is also equipped with the strong PT7 promoter (and an efficient ribosome binding site) more proximal to the MtCM gene, in tandem to Ptet (31Roderer K. Neuenschwander M. Codoni G. Sasso S. Gamper M. Kast P. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.PLoS ONE. 2014; 9 (25551646)e11623410.1371/journal.pone.0116234Crossref PubMed Scopus (6) Google Scholar). PT7-directed gene overexpression for biochemical studies just required retransformation of candidate plasmids into strain KA12 containing plasmid pT7POLTS, which carries the gene for the PT7-specific T7 RNA polymerase (31Roderer K. Neuenschwander M. Codoni G. Sasso S. Gamper M. Kast P. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.PLoS ONE. 2014; 9 (25551646)e11623410.1371/journal.pone.0116234Crossref PubMed Scopus (6) Google Scholar).Table 1Comparison of the catalytic parameters of His-tagged and untagged evolved MtCM variantsCycleProteinMutationsaPD/PDAM stands for T52P, V55D, R87P, L88D, G89A, and H90M replacements.kcatbCM activity was determined in 50 mm potassium phosphate buffer, pH 7.5, containing 0.1 mg/ml BSA. The disappearance of chorismate was monitored at 310 nm (30 °C). Kinetic parameters were derived by fitting specific initial velocities to the Michaelis-Menten equation (raw data for representative examples are shown in Fig. S6). kcat is calculated per active site. Substrate concentration was varied between 20 and 2000 µm; nd, not determined because substrate saturation could not be achieved due to high chorismate absorption above 2 mm. Mean and standard deviation (σn−1) were derived from at least two independent biological replicates. (s−1)KmbCM activity was determined in 50 mm potassium phosphate buffer, pH 7.5, containing 0.1 mg/ml BSA. The disappearance of chorismate was monitored at 310 nm (30 °C). Kinetic parameters were derived by fitting specific initial velocities to the Michaelis-Menten equation (raw data for representative examples are shown in Fig. S6). kcat is calculated per active site. Substrate concentration was varied between 20 and 2000 µm; nd, not determined because substrate saturation could not be achieved due to high chorismate absorption above 2 mm. Mean and standard deviation (σn−1) were derived from at least two independent biological replicates. (μm)kcat/KmcValues obtained from error propagation. ×104 (m−1 s−1)TmeThe melting temperature was determined using CD spectroscopy. Raw data and the fitting equation for representative MtCM variants are shown in Fig. S7. (°C)MtCMfData from the literature (11).2.0 ± 0.11140 ± 900.175 ± 0.00974 ± 0MtCMfData from the literature (11). + MtDSgEnzyme variants containing an N-terminal His6-tag.8.1 ± 1.934 ± 324 ± 6-0H6-MtCMgEnzyme variants containing an N-terminal His6-tag.-nd>17000.094 ± 0.006dValue obtained from averaged initial velocities of respective Michaelis-Menten plots.75 ± 0I3p3gEnzyme variants containing an N-terminal His6-tag.PD12 ± 0570 ± 172.1 ± 0.181 ± 1II3p5gEnzyme variants containing an N-terminal His6-tag.PD/PDAM31 ± 2380 ± 208.3 ± 0.882 ± 0IIIre4.7s11gEnzyme variants containing an N-terminal His6-tag.(= s11)PD/PDAM, V62I, D72V23 ± 372 ± 733 ± 5>88IVs10es4.15gEnzyme variants containing an N-terminal His6-tag.(= s4.15)PD/PDAM, V62I, D72V, V11L, D15V, K40Q14 ± 231 ± 745 ± 1182 ± 1N-re4.7s11(= N-s11)PD/PDAM, V62I, D72V28 ± 262 ± 1745 ± 1388 ± 1N-s10es4.15(= N-s4.15)PD/PDAM, V62I, D72V, V11L, D15V, K40Q20 ± 443 ± 747 ± 1183 ± 1a PD/PDAM stands for T52P, V55D, R87P, L88D, G89A, and H90M replacements.b CM activity was determined in 50 mm potassium phosphate buffer, pH 7.5, containing 0.1 mg/ml BSA. The disappearance of chorismate was monitored at 310 nm (30 °C). Kinetic parameters were derived by fitting specific initial velocities to the Michaelis-Menten equation (raw data for representative examples are shown in Fig. S6). kcat is calculated per active site. Substrate concentration was varied between 20 and 2000 µm; nd, not determined because substrate saturation could not be achieved due to high chorismate absorption above 2 mm. Mean and standard deviation (σn−1) were derived from at least two independent biological replicates.c Values obtained from error propagation.d Value obtained from averaged initial velocities of respective Michaelis-Menten plots.e The melting temperature was determined using CD spectroscopy. Raw data and the fitting equation for representative MtCM variants are shown in Fig. S7.f Data from the literature (11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar).g Enzyme variants containing an N-terminal His6-tag. Open table in a new tab During a first evolutionary cycle (cycle I), the flexible loop between helices H1 and H2 of H6-MtCM was targeted by cassette mutagenesis (Fig. 3). This loop, encompassing residues 50 to 55, underwent significant structural changes upon interaction with MtDS resulting in MtCM activation (11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar) (Fig. 1C). Residues Thr52, Arg53, Leu54, and Val55 from the H1-H2 loop were randomized via NNK (N = A/C/G/T, K = G/T) codons, and the resulting gene library was transformed into KA12/pKIMP-UAUC to select for active catalysts (Fig. 2). Sequence analysis of the mutant H6-MtCM genes from 34 transformants growing after 4 days at 30 °C on M9c minimal medium revealed a remarkable pattern. With the exception of two clones showing Cys, Val55 was replaced by an aspartate residue in all selected MtCM variants (Fig. 4A and Fig. S1). Positions 53 (Arg) and 54 (Leu) of MtCM were somewhat more variable and tolerated chemically similar replacements. Position 52 (Thr) showed a slight preference for both Pro and Ser instead of the WT Thr in MtCM. Variant PHS08-3p3 (henceforth called 3p3), which combines the two substitutions T52P and V55D, has a 22-fold higher catalytic efficiency (kcat/Km = 2.1 × 104m−1 s−1) than H6-MtCM.Figure 4Selected MtCM sequences from evolutionary cycles I and II. A, results from cycle I of directed evolution. Shown is the amino acid distribution at the randomized positions as derived from sequencing of 34 TRLV library members growing on selective M9c plates. B, cycle II sequencing data. Included are 52 variants selected on M9c+pFPhe from libraries CT7, CT-LGH, and CT-RLGH. Circle size and color correlate with the frequency of individual encoded residues with the color code shown below the panels. Amino acids are listed with their one-letter abbreviation. The percentage of a particular residue at each randomized position is given within the circle, and the absolute number of codons considered in the compilations is indicated in parentheses next to the WT residue listed on the left for each sampled position.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For evolutionary cycle II (Fig. 3), 3p3 was used as a template to randomize C-terminal residues, which are known to be important for the catalytic machinery of MtCM (Gly84, Arg85, and Gly86) or MtDS-mediated activation of MtCM (Arg87, Leu88, Gly89, and His90) (11Sasso S. Ökvist M. Roderer K. Gamper M. Codoni G. Krengel U. Kast P. Structure and function of a complex between chorismate mutase and DAHP synthase: efficiency boost for the junior partner.EMBO J. 2009; 28 (19556970): 2128-214210.1038/emboj.2009.165Crossref PubMed Scopus (37) Google Scholar, 31Roderer K. Neuenschwander M. Codoni G. Sasso S. Gamper M. Kast P. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.PLoS ONE. 2014; 9 (25551646)e11623410.1371/journal.pone.0116234Crossref PubMed Scopus (6) Google Scholar). The three independent libraries PD/LGH, PD/RLGH, and PD/CT7 randomized three, four, and seven positions in the C-terminal region, respectively. Thereby, the codon format NNN allowed for all three translational stop codons to probe for truncated active enzymes (Fig. 3). Indeed, the results suggest that the two to four most C-terminal residues are dispensable in some highly functional variants (Fig. 4B). Interestingly, only Cys, Ser, or Gly appear to be allowed at position 84 (Gly in WT MtCM), and the WT residues Arg85 and Gly86 clearly dominated at the corresponding positions (Fig. 4B and Fig. S1). The best of the characterized catalysts (PHS10-3p5; 3p5) from the PD/RLGH library contained the substitutions R87P, L88D, G89A, and H90M at the C terminus, in addition t" @default.
• W3092443721 created "2020-10-15" @default.
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• W3092443721 date "2020-12-01" @default.
• W3092443721 modified "2023-10-09" @default.
• W3092443721 title "Evolving the naturally compromised chorismate mutase from Mycobacterium tuberculosis to top performance" @default.
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• W3092443721 doi "https://doi.org/10.1074/jbc.ra120.014924" @default.
• W3092443721 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7762937" @default.
• W3092443721 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33453995" @default.
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