FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2025553933> ?p ?o ?g. }

• W2025553933 endingPage "7169" @default.
• W2025553933 startingPage "7162" @default.
• W2025553933 abstract "YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents. YdiB and its paralog AroE are members of the quinate/shikimate 5-dehdrogenase family. Enzymes from this family function in the shikimate pathway that is essential for survival of microorganisms and plants and represent potential drug targets. Recent YdiB and AroE crystal structures revealed the presence of a NAD(P)-binding and a catalytic domain. We carried out site-directed mutagenesis of 8 putative active site residues in YdiB from Escherichia coli and analyzed structural and kinetic properties of the mutant enzymes. Our data indicate critical roles for an invariant lysine and aspartate residue in substrate binding and allowed us to differentiate between two previously proposed models for the binding of the substrate in the active site. Comparison of several YdiB and AroE structures led us to conclude that, upon cofactor binding and domain closure, the 2 identified binding residues are repositioned to bind to the substrate. Although the lysine residue contributes to some extent to the stabilization of the transition state, we did not identify any residue as catalytically essential. This indicates that catalysis does not operate through a general acid-base mechanism, as thought originally. Our improved understanding of the medically and agriculturally important quinate/shikimate 5-dehydrogenase family at the molecular level may prove useful in the development of novel herbicides and antimicrobial agents. The shikimate pathway of prokaryotes, fungi, plants, and apicomplexa is essential for survival. The main route of this pathway leads to the biosynthesis of chorismate, the precursor of essential aromatic compounds including vitamins and amino acids (Fig. 1) (1Herrmann K.M. Weaver L.M. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 473-503Crossref PubMed Scopus (868) Google Scholar, 2Knaggs A.R. Nat. Prod. Rep. 2003; 20: 119-136Crossref PubMed Scopus (134) Google Scholar). The successful targeting of the shikimate pathway in crop plants by glyphosate has spurred further efforts in herbicide development (3Coggins J.R. Abell C. Evans L.B. Frederickson M. Robinson D.A. Roszak A.W. Lapthorn A.P. Biochem. Soc. Trans. 2003; 31: 548-552Crossref PubMed Scopus (81) Google Scholar). More recently, the occurrence of herbicide-resistant weeds (4Baerson S.R. Rodriguez D.J. Tran M. Feng Y. Biest N.A. Dill G.M. Plant Physiol. 2002; 129: 1265-1275Crossref PubMed Scopus (271) Google Scholar) has led to the development of transgenic crops with increased glyphosate tolerance (5Carpenter J. Gianessi L. Science. 2000; 287: 803-804Crossref PubMed Google Scholar, 6Castle L.A. Siehl D.L. Gorton R. Patten P.A. Chen Y.H. Bertain S. Cho H.J. Duck N. Wong J. Liu D. Lassner M.W. Science. 2004; 304: 1151-1154Crossref PubMed Scopus (239) Google Scholar). In bacteria, the shikimate pathway has been subject to metabolic engineering, aimed at the possible industrial production of high value hydroaromatic compounds (7Kra ̈mer M. Bongaerts J. Bovenberg R. Kremer S. Mu ̈ller U. Orf S. Wubbolts M. Raeven L. Metab. Eng. 2003; 5: 277-283Crossref PubMed Scopus (188) Google Scholar). Lastly, their absence in metazoans makes the enzymes of the shikimate pathway potential targets for novel antimicrobial agents (3Coggins J.R. Abell C. Evans L.B. Frederickson M. Robinson D.A. Roszak A.W. Lapthorn A.P. Biochem. Soc. Trans. 2003; 31: 548-552Crossref PubMed Scopus (81) Google Scholar, 8Payne D.J. Wallis N.G. Gentry D.R. Rosenberg M. Curr. Opin. Drug Discovery Dev. 2000; 3: 177-190PubMed Google Scholar, 9Parish T. Stoker N.G. Microbiology (Reading). 2002; 148: 3069-3077Crossref PubMed Scopus (166) Google Scholar, 10Davies G.M. Barrett-Bee K.J. Jude D.A. Lehan M. Nichols W.W. Pinder P.E. Thain J.L. Watkins W.J. Wilson R.G. Antimicrob. Agents Chemother. 1994; 38: 403-406Crossref PubMed Scopus (107) Google Scholar). With a view to the design of inhibitors of the shikimate pathway, the understanding at the molecular level of enzymes involved in this pathway has received much attention over the last 25 years (3Coggins J.R. Abell C. Evans L.B. Frederickson M. Robinson D.A. Roszak A.W. Lapthorn A.P. Biochem. Soc. Trans. 2003; 31: 548-552Crossref PubMed Scopus (81) Google Scholar, 11Brown K.A. Carpenter E.P. Watson K.A. Coggins J.R. Hawkins A.R. Koch M.H. Svergun D.I. Biochem. Soc. Trans. 2003; 31: 543-547Crossref PubMed Google Scholar). Step 4 of the pathway, the reversible NADPH-dependent reduction of 3-dehydroshikimate to shikimate (Fig. 2) is catalyzed by the enzyme shikimate dehydrogenase (EC 1.1.1.25), a member of the quinate/shikimate 5-dehdrogenase family. In addition to the widely distributed bacterial NADP-dependent shikimate dehydrogenase AroE, Escherichia coli, Salmonella typhimurium, Streptococcus pneumoniae, and Haemophilus influenzae also possess a paralogous enzyme, YdiB. YdiB from E. coli is a dual specificity quinate/shikimate NAD-dependent dehydrogenase, and its possible evolution and metabolic role have been discussed recently (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Although the involvement of AroE in the shikimate pathway is well established for E. coli and S. typhimurium (13Pittard J. Wallace B.J. J. Bacteriol. 1966; 91: 1494-1508Crossref PubMed Google Scholar), the actual biological function of YdiB remains unclear, nor is it known whether 3-dehydroshikimate or quinate represents the natural substrate of YdiB. Nevertheless, the YdiB enzymes from S. pneumoniae and H. influenzae were recently shown to be essential for in vitro growth of these human pathogens (14Zalacain M. Biswas S. Ingraham K.A. Ambrad J. Bryant A. Chalker A.F. Iordanescu S. Fan J. Fan F. Lunsford R.D. O'Dwyer K. Palmer L.M. So C. Sylvester D. Volker C. Warren P. McDevitt D. Brown J.R. Holmes D.J. Burnham M.K. J. Mol. Microbiol. Biotechnol. 2004; 6: 109-126Crossref Scopus (75) Google Scholar). A similar occurrence of two family members is also found in filamentous fungi. In addition to a NADP-dependent shikimate dehydrogenase, they possess a related enzyme, quinate dehydrogenase (EC 1.1.1.24), that catalyzes the NAD-dependent oxidation of quinate. This is the first step of the quinate pathway (Fig. 1), which affords growth on quinate as a carbon source (15Hawkins A.R. Lamb H.K. Moore J.D. Charles I.G. Roberts C.F. J. Gen. Microbiol. 1993; 139: 2891-2899Crossref PubMed Scopus (80) Google Scholar). In fungi (16Hua S. Guo T. Gough J. Sun Z. J. Mol. Biol. 2002; 320: 713-719Crossref PubMed Scopus (9) Google Scholar) and the apicomplexan parasite Toxoplasma gondii (17Campbell S.A. Richards T.A. Mui E.J. Samuel B.U. Coggins J.R. McLeod R. Roberts C.W. Int. J. Parasitol. 2004; 34: 5-13Crossref PubMed Scopus (67) Google Scholar), the NADP-dependent shikimate dehydrogenase constitutes the C-terminal module of a penta-functional polypeptide, which combines the enzymatic activities for steps 2–6 of the shikimate pathway (Fig. 1). In plants (18Schmid J. Amrhein N. Phytochemistry. 1995; 39: 737-749Crossref Scopus (170) Google Scholar) and bacteria from the genus Chlamydia, this enzyme forms the C-terminal module of a bifunctional polypeptide catalyzing steps 3 and 4. Most bacterial shikimate dehydrogenases, and the fungal quinate-oxidizing dehydrogenases, on the other hand, represent monofunctional enzymes of 29–36 kDa. Few studies have been performed on the kinetic or chemical mechanism of catalysis for this family of enzymes. For the shikimate dehydrogenase from Pisum sativum, it has been shown that the kinetic mechanism is ordered Bi Bi in both directions, with the cofactor adding first (19Balinsky D. Dennis A.W. Cleland W.W. Biochemistry. 1971; 10: 1947-1952Crossref PubMed Scopus (29) Google Scholar). As additionally demonstrated for the shikimate dehydrogenase from E. coli (20Dansette P. Azerad R. Biochimie (Paris). 1974; 56: 751-755Crossref PubMed Scopus (11) Google Scholar), the mechanism involves the stereoselective transfer of hydrogen between the A side of NADPH and the substrate (19Balinsky D. Dennis A.W. Cleland W.W. Biochemistry. 1971; 10: 1947-1952Crossref PubMed Scopus (29) Google Scholar). Recently, the crystal structures of AroE from E. coli, Methanococcus jannaschii, and H. influenzae (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Padyana A.K. Burley S.K. Structure. 2003; 11: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 22Ye S. von Delft F. Brooun A. Knuth M.W. Swanson R.V. McRee D.E. J. Bacteriol. 2003; 185: 4144-4151Crossref PubMed Scopus (44) Google Scholar) and of YdiB from E. coli (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) were solved in complex with NADP and NAD, respectively. Structures of apoenzymes are also available for H. influenzae AroE (22Ye S. von Delft F. Brooun A. Knuth M.W. Swanson R.V. McRee D.E. J. Bacteriol. 2003; 185: 4144-4151Crossref PubMed Scopus (44) Google Scholar) and YdiB. 1S. Korolev, O. Koroleva, T. Zarembinski, F. Collart, and A. Joachimiak, unpublished results. 1S. Korolev, O. Koroleva, T. Zarembinski, F. Collart, and A. Joachimiak, unpublished results. All of these structures reveal a common fold comprising two domains separated by a cleft. Although AroE from E. coli and AroE and YdiB from H. influenzae exist as monomers (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Ye S. von Delft F. Brooun A. Knuth M.W. Swanson R.V. McRee D.E. J. Bacteriol. 2003; 185: 4144-4151Crossref PubMed Scopus (44) Google Scholar), YdiB from E. coli (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) and AroE from M. jannaschii (21Padyana A.K. Burley S.K. Structure. 2003; 11: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) both homodimerize via their N-terminal domains. This portion of the protein possesses a unique α-β-α sandwich motif, which also includes an α-helical hairpin structure at the C terminus of the protein and is further referred to as domain 1. The intervening sequence, domain 2, forms a Rossmann fold, to which the dinucleotide cofactor is bound with the A side of the nicotinamide ring facing the interdomain cleft (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Padyana A.K. Burley S.K. Structure. 2003; 11: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). A direct comparison of the cofactor complexes for the two E. coli enzymes AroE and YdiB by Michel et al. (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) revealed structural differences in their nucleotide-binding motifs, which likely account for the 10-fold higher affinity of YdiB for NAD than for NADP. In contrast to E. coli YdiB, AroE enzymes are generally NADP-specific. Despite the recent availability of several crystal structures for enzyme-cofactor complexes, as well as for apoenzymes of quinate/shikimate 5-dehydrogenases, no structural data on substrate binding are yet available, and so the nature of the active site in this agriculturally and medically important enzyme family has remained ambiguous. The active site is identified by the position of the nicotinamide moiety and appears to be defined by a conserved cluster of residues from domain 1 (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 21Padyana A.K. Burley S.K. Structure. 2003; 11: 1005-1013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). We carried out mutational analyses of 8 putative active site residues from domain 1 in YdiB from E. coli, exhausting all apparent candidate residues for catalysis. A conserved asparagine proved to be essential for proper folding. In addition to structural characterization and steady state kinetics, comparison of the mutated residues in different YdiB and AroE crystal structures supports roles for an invariant aspartate and lysine residue in substrate binding. Surprisingly, none of the mutated residues was essential for catalysis, suggesting that the primary catalytic mechanism does not involve general acid-base catalysis. Preparation and Characterization of YdiB Mutants—Site-directed mutagenesis was performed on the plasmid containing the ydiB gene (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) using the QuikChange™ XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). Forward versions of the mutagenic primers are listed in Table I. The sequences of the mutant ydiB genes were confirmed by DNA sequencing. YdiB enzymes were overexpressed and purified as described previously for wild-type protein (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories) based on the original Bradford assay (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211983) Google Scholar) with bovine serum albumin as the standard. Mass spectra were recorded using an Agilent 1100 Series liquid chromatograph/mass selective detector instrument (Agilent Technologies, Mississauga, Ontario, Canada) in electrospray ionization mode and analyzed using Agilent ChemStation software (version A.09.01). A sample of purified YdiB protein (0.1–0.2 mg/ml) was diluted 1:100 (v/v) in 10% (v/v) acetonitrile, 0.1% (v/v) formic acid prior to injection. For native PAGE, proteins were analyzed using a 12% (w/v) polyacrylamide gel (375 mm Tris/HCl, pH 8.8) containing 8.7% (v/v) glycerol. After the addition of one equal volume of 2× loading buffer (125 mm Tris/HCl, pH 6.8, 20% (v/v) glycerol, 10 μg/ml bromphenol blue), 3 μg of protein were loaded per lane. Electrophoresis was performed at 125 mA for 3 h with cooling on ice. Gels were stained with 0.1% (w/v) Coomassie Blue R-350. Dynamic light scattering and analytical gel filtration were performed as described previously (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In brief, for dynamic light scattering measurements, 50 μl of protein (0.3 mg/ml) were cleared by centrifugation and transferred to the 96-well plate. Analytical gel filtration was performed by applying 180 μg of protein to the column at a flow rate of 0.5–0.6 ml/min. CD spectra were recorded using a model J-710 spectropolarimeter from Jasco Inc. (Easton, MD) with an N2 flow rate of 5 liters/min and were analyzed using the Jasco J-700 software for Windows v1.10.00. Protein samples (200 μl) at a concentration of ∼0.6–0.8 mg/ml in buffer (50 mm Tris/HCl, pH 7.5, 200 mm NaCl, 5% (v/v) glycerol) were loaded into a CD circular quartz cuvette (path length = 0.05 cm). For each sample, five scans were acquired at a scan speed of 50 nm/min, with a bandwidth of 1.0 nm and a step resolution of 0.2 nm in the wavelength range of 200–250 nm. The scans were subsequently averaged. Spectra of buffer alone were recorded accordingly and subtracted from the protein spectra.Table IForward versions of mutagenic primers, and sequence conservation of the mutated residues in the quinate/shikimate 5-dehydrogenase familyMutantOligonucleotide sequenceaAltered nucleotides, that introduced the desired mutations, are underlined. Additional silent mutations, introducing restriction enzyme cleavage sites for screening purposes, are printed in italicsDegree of residue conservationbThe degree of conservation for the mutated residue in E. coli YdiB among ∼75 members of the quinate/shikimate 5-dehydrogenase family, as aligned in the NCBI Conserved Domain Database4 (28), is indicatedS22A5′-TATCCGCCACAGTTTGGCGCCCGAAATGCAGAATAAAG-3′76%Y39F5′-AATAAAGCCTTAGAAAAAGCGGGATTGCCATTTACCTTTATGGCATTCGAAGTGGATAACGATAGCTTTCCTGG-3′85%S67A5′-ATGCGCGGAACTGGTGTCGCGATGCCGAACAA-3′21%cThreonine, not serine is the prevailing amino acid at position 67, with a conservation level of 40%K71G5′-CTGGTGTATCGATGCCGAATGGACAACTGGCGTGTGAATATG-3′100%N92A5′-TGCCAAACTGGTGGGGGCCATCGCCACCATCGTTAATGATGATGGCTATCT-3′99%T106A5′-CGTGGCTATAACGCCGACGGCACGG-3′99%D107A5′-CTATCTGCGTGGCTATAACACCGCCGGCACGGGCCATATTCGCGC-3′100%Q262A5′-ATGGATACGGCATGTTGTTGTGGGCCGGCGCTGAACAGTTCACATTATGGAC-3′99%a Altered nucleotides, that introduced the desired mutations, are underlined. Additional silent mutations, introducing restriction enzyme cleavage sites for screening purposes, are printed in italicsb The degree of conservation for the mutated residue in E. coli YdiB among ∼75 members of the quinate/shikimate 5-dehydrogenase family, as aligned in the NCBI Conserved Domain Database4 (28Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler G.H. Mazumder R. Nikolskaya A.N. Panchenko A.R. Rao B.S. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (648) Google Scholar), is indicatedc Threonine, not serine is the prevailing amino acid at position 67, with a conservation level of 40% Open table in a new tab Steady State Kinetics—Enzymatic activities were measured in 100 mm Tris/HCl, pH 9, in the reverse direction as described previously (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) with the following modifications. Determinations were set up in quadruplicate in 96-well plates using a multichannel pipette for synchronous mixing of the reagents. To determine the substrate- and cofactor-dependent kinetic properties of YdiB variants, we prepared 11 different concentrations each of shikimate, quinate, and NAD by one-in-one dilutions (v/v) of a 4 or 20 mm substrate stock solution and a 4 mm NAD stock solution, respectively. Per well, 180 μl of substrate or cofactor were added to 10 μl of enzyme, and the reactions were started by adding 20 μlofa40mm solution of NAD or substrate (i.e. shikimate or quinate), respectively. Initial velocities of enzyme activity were determined by recording NADH absorbance at 340 nm in a 96-well plate reader. Each reading was calibrated by including a series of eight one-in-one dilutions (v/v) of reduced cofactor in a volume of 210 μl/well, starting from 0.4 mm NADH. Kinetic data were evaluated by non-linear regression analysis with the Michaelis-Menten equation (v = Vmax × [S]/(KM + [S])), using the SigmaPlot software (SPSS Science, Chicago, IL). The catalytic constant, kcat, was calculated using the equation Vmax = kcat × [E], where [E] = total enzyme concentration. Structure Comparison—Structural models 2The atomic coordinates for the crystal structures of YdiB from E. coli and H. influenzae and AroE from E. coli, H. influenzae, and M. jannaschii were obtained from the Protein Data Bank (http://www.rcsb.org/pdb) under PDB numbers 1O9B/1NPD, 1NPY, 1NYT, 1P74/1P77, and 1NVT, respectively (25Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26227) Google Scholar). Although 1NPY and 1P74 represent apoenzymes, all remaining models feature complexes with the NAD(P) cofactor. for YdiB from E. coli and H. influenzae and for AroE from E. coli, H. influenzae, and M. jannaschii were superimposed, and root mean square deviation calculations were carried out using the Swiss-PDBviewer (26Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9399) Google Scholar). Structural Characterization of YdiB Mutants—The structure of E. coli YdiB has been determined independently by two groups (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Although one of the E. coli YdiB models (PDB 3The abbreviation used is: PDB, Protein Data Bank. number 1NPD) includes all 288 residues of the protein (23Benach J. Lee I. Edstrom W. Kuzin A.P. Chiang Y. Acton T.B. Montelione G.T. Hunt J.F. J. Biol. Chem. 2003; 278: 19176-19182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), the other (PDB number 1O9B) includes residues 7–286 (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the current work, we made use of the same YdiB construct and YdiB purification protocol as described in the latter report (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To determine whether the purified enzyme was truncated, we analyzed the wild-type protein by mass spectroscopy. Liquid chromatograph/mass selective detector analysis gave a mass for YdiB of 31,361 Da, as compared with a calculated mass of 31,371 Da, which agrees within the expected mass error of about 0.02%. This mass includes the additional Gly-Ser residues that remain at the N terminus after thrombin cleavage with protein expressed using the pGEX-4T1 vector (12Michel G. Roszak A.W. Sauve V. Maclean J. Matte A. Coggins J.R. Cygler M. Lapthorn A.J. J. Biol. Chem. 2003; 278: 19463-19472Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the following, we will refer to residues according to E. coli YdiB numbering. Where specified otherwise, E. coli YdiB numbering will be referenced in parentheses. Sequence alignments of quinate/shikimate 5-dehydrogenase family members from Pfam (27Bateman A. Coin L. Durbin R. Finn R.D. Hollich V. Griffiths-Jones S. Khanna A. Marshall M. Moxon S. Sonnhammer E.L.L. Studholme D.J. Yeats C. Eddy S.R. Nucleic Acids Res. 2004; 32: D138-D141Crossref PubMed Google Scholar) and the NCBI Conserved Domain Database 4The respective sequence alignments of quinate/shikimate 5-dehydrogenase family members can be accessed through Pfam (http://www.sanger.ac.uk/Software/Pfam/) under accession number Pf01488 and through the NCBI Protein Database (http://www.ncbi.nlm.nih.gov) under NCBI accession number COG0169. (28Marchler-Bauer A. Anderson J.B. DeWeese-Scott C. Fedorova N.D. Geer L.Y. He S. Hurwitz D.I. Jackson J.D. Jacobs A.R. Lanczycki C.J. Liebert C.A. Liu C. Madej T. Marchler G.H. Mazumder R. Nikolskaya A.N. Panchenko A.R. Rao B.S. Shoemaker B.A. Simonyan V. Song J.S. Thiessen P.A. Vasudevan S. Wang Y. Yamashita R.A. Yin J.J. Bryant S.H. Nucleic Acids Res. 2003; 31: 383-387Crossref PubMed Scopus (648) Google Scholar) indicate that the residues Gly63, Ser/Thr67, Pro69, Lys71, Asn92, Asp107, Gly131, Gly133, Gly134, Gly255, and Gln262 are likely fully conserved. In addition, Asn105 and Arg156 are also conserved in nearly all sequences. The cluster of glycines (at positions 131, 133, and 134) is part of the P-loop that interacts with the phosphates of the NAD(P) cofactor, and the carbonyl group of Gly255 hydrogen-bonds to the amide group of the nicotinamide ring. We have concentrated on the highly conserved residues Ser67, Lys71, Asn92, Asp107, and Gln262 in the vicinity of the putative substrate-binding site as the most likely candidates for residues essential for catalysis. Additionally, we selected the partially conserved residues Ser22, Tyr39, and Thr106 as potential substrate-binding residues (Table I, Fig. 3). We have not attempted to mutate Arg156 as it is remote from the catalytic site and stacks with the adenine ring of NAD. Similarly, Asn105 appears to be shielded from the active site by Asp107 and likely plays a structural role. The mutants S22A, Y39F, S67A, K71G, N92A, T106A, D107A, and Q262A were expressed and, with the exception of N92A, purified to apparent homogeneity as assessed by SDS-PAGE (not shown). The N92A mutant protein appeared more sensitive to proteolytic cleavage than wild-type YdiB or any of the other mutants and was not isolated in a pure form. To assess the effects of the various mutations on YdiB structure, we characterized the conformational integrity of the purified proteins using several methods. Native PAGE showed well focused bands for the wild-type protein, as well as for the S22A, K71G, Q262A, Y39F, and S67A mutants, and less-focused bands for the D107A and T106A mutants (Fig. 4A). The N92A mutant appeared as a smear. Dynamic light scattering measurements on the various purified proteins were mostly in agreement with the results from native PAGE, with the N92A mutant being polydisperse and yielding an apparent molecular mass of 300 kDa (not shown). Consistent with the results from native PAGE, the general shape of the CD spectra for wild-type enzyme and the S67A and K71G mutants were similar, whereas the alanine mutations of Thr106 and Asp107 caused apparent shifts (Fig. 4B). The N92A mutant exhibited a spectrum that deviated significantly from either wild-type YdiB or the other YdiB mutants. In analytical gel filtration experiments, wild-type YdiB exhibited an apparent mass of ∼57 kDa (calculated mass for the recombinant dimer: 62.7 kDa). In addition, the wild-type contained a very small amount (0.2%) of a species with an apparent mass of ∼33 kDa (likely monomeric form) and some protein that eluted in the void volume of the column. About 65% of the N92A mutant protein appeared in the void volume and was likely aggregated, with the remainder eluting at about 33 kDa. Although the T106A mutant also showed considerable amounts of apparent aggregate (12%) and a 33-kDa species (1.8%), it largely eluted as a 57-kDa species (likely dimeric form). The D107A profile was most similar to that of the wild type. Taken together, these data suggested that the N92A protein was improperly folded. The protein was partly degraded, likely by trace amounts of proteolytic enzymes. We assume that the fragments aggregated into larger species. The Asn92 residue is located at a β-bulge, at the N-terminal end of strand β4 within the 6-stranded mixed β-sheet of domain 1 (Fig. 3), and participates in a hydrogen-bonding network through its side chain and main chain atoms (12Michel G. Rosz" @default.
• W2025553933 created "2016-06-24" @default.
• W2025553933 creator A5005759921 @default.
• W2025553933 creator A5010480312 @default.
• W2025553933 creator A5015099789 @default.
• W2025553933 creator A5025328829 @default.
• W2025553933 creator A5082328626 @default.
• W2025553933 creator A5090164097 @default.
• W2025553933 date "2005-02-01" @default.
• W2025553933 modified "2023-09-30" @default.
• W2025553933 title "Site-directed Mutagenesis of the Active Site Region in the Quinate/Shikimate 5-Dehydrogenase YdiB of Escherichia coli" @default.
• W2025553933 cites W1501364613 @default.
• W2025553933 cites W1502488094 @default.
• W2025553933 cites W1523682304 @default.
• W2025553933 cites W1606067198 @default.
• W2025553933 cites W1681177208 @default.
• W2025553933 cites W1947762817 @default.
• W2025553933 cites W1981251699 @default.
• W2025553933 cites W1985829653 @default.
• W2025553933 cites W1989631075 @default.
• W2025553933 cites W1998526948 @default.
• W2025553933 cites W2008428704 @default.
• W2025553933 cites W2008603784 @default.
• W2025553933 cites W2015642465 @default.
• W2025553933 cites W2026334274 @default.
• W2025553933 cites W2033051115 @default.
• W2025553933 cites W2044196573 @default.
• W2025553933 cites W2046883007 @default.
• W2025553933 cites W2056245360 @default.
• W2025553933 cites W2078763795 @default.
• W2025553933 cites W2079937125 @default.
• W2025553933 cites W2091368419 @default.
• W2025553933 cites W2096235134 @default.
• W2025553933 cites W2103797225 @default.
• W2025553933 cites W2106857639 @default.
• W2025553933 cites W2107959468 @default.
• W2025553933 cites W2111458535 @default.
• W2025553933 cites W2111675603 @default.
• W2025553933 cites W2117397036 @default.
• W2025553933 cites W2122876047 @default.
• W2025553933 cites W2130479394 @default.
• W2025553933 cites W2147045968 @default.
• W2025553933 cites W2165500111 @default.
• W2025553933 cites W2169168434 @default.
• W2025553933 cites W2418835718 @default.
• W2025553933 cites W4210400672 @default.
• W2025553933 cites W4293247451 @default.
• W2025553933 doi "https://doi.org/10.1074/jbc.m412028200" @default.
• W2025553933 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15596430" @default.
• W2025553933 hasPublicationYear "2005" @default.
• W2025553933 type Work @default.
• W2025553933 sameAs 2025553933 @default.
• W2025553933 citedByCount "26" @default.
• W2025553933 countsByYear W20255539332012 @default.
• W2025553933 countsByYear W20255539332013 @default.
• W2025553933 countsByYear W20255539332014 @default.
• W2025553933 countsByYear W20255539332015 @default.
• W2025553933 countsByYear W20255539332017 @default.
• W2025553933 countsByYear W20255539332018 @default.
• W2025553933 countsByYear W20255539332020 @default.
• W2025553933 countsByYear W20255539332021 @default.
• W2025553933 countsByYear W20255539332022 @default.
• W2025553933 countsByYear W20255539332023 @default.
• W2025553933 crossrefType "journal-article" @default.
• W2025553933 hasAuthorship W2025553933A5005759921 @default.
• W2025553933 hasAuthorship W2025553933A5010480312 @default.
• W2025553933 hasAuthorship W2025553933A5015099789 @default.
• W2025553933 hasAuthorship W2025553933A5025328829 @default.
• W2025553933 hasAuthorship W2025553933A5082328626 @default.
• W2025553933 hasAuthorship W2025553933A5090164097 @default.
• W2025553933 hasBestOaLocation W20255539331 @default.
• W2025553933 hasConcept C103408237 @default.
• W2025553933 hasConcept C104317684 @default.
• W2025553933 hasConcept C143065580 @default.
• W2025553933 hasConcept C16318435 @default.
• W2025553933 hasConcept C181199279 @default.
• W2025553933 hasConcept C185592680 @default.
• W2025553933 hasConcept C2778255928 @default.
• W2025553933 hasConcept C501734568 @default.
• W2025553933 hasConcept C547475151 @default.
• W2025553933 hasConcept C553450214 @default.
• W2025553933 hasConcept C55493867 @default.
• W2025553933 hasConcept C86803240 @default.
• W2025553933 hasConceptScore W2025553933C103408237 @default.
• W2025553933 hasConceptScore W2025553933C104317684 @default.
• W2025553933 hasConceptScore W2025553933C143065580 @default.
• W2025553933 hasConceptScore W2025553933C16318435 @default.
• W2025553933 hasConceptScore W2025553933C181199279 @default.
• W2025553933 hasConceptScore W2025553933C185592680 @default.
• W2025553933 hasConceptScore W2025553933C2778255928 @default.
• W2025553933 hasConceptScore W2025553933C501734568 @default.
• W2025553933 hasConceptScore W2025553933C547475151 @default.
• W2025553933 hasConceptScore W2025553933C553450214 @default.
• W2025553933 hasConceptScore W2025553933C55493867 @default.
• W2025553933 hasConceptScore W2025553933C86803240 @default.
• W2025553933 hasIssue "8" @default.
• W2025553933 hasLocation W20255539331 @default.
• W2025553933 hasOpenAccess W2025553933 @default.

• next