FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2144304328> ?p ?o ?g. }

• W2144304328 endingPage "11038" @default.
• W2144304328 startingPage "11028" @default.
• W2144304328 abstract "The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins. The structure and biochemical function of the hot dog-fold thioesterase PaaI operative in the aerobic phenylacetate degradation pathway are examined. PaaI showed modest activity with phenylacetyl-coenzyme A, suggestive of a role in coenzyme A release from this pathway intermediate in the event of limiting downstream pathway enzymes. Minimal activity was observed with aliphatic acyl-coenzyme A thioesters, which ruled out PaaI function in the lower phenylacetate pathway. PaaI was most active with ring-hydroxylated phenylacetyl-coenzyme A thioesters. The x-ray crystal structure of the Escherichia coli thioesterase is reported and analyzed to define the structural basis of substrate recognition and catalysis. The contributions of catalytic and substrate binding residues, thus, identified were examined through steady-state kinetic analysis of site-directed mutant proteins. Aromatic compounds serve as a rich source of carbon and energy for a wide variety of microorganisms and plants (1Ramos J.L. Gonzalez-Perez M.M. Caballero A. van Dillewijn P. Curr. Opin. Biotechnol. 2005; 16: 275-281Crossref PubMed Scopus (83) Google Scholar). The enzymes that make up the aromatic catabolic pathways are useful for bioremediation of environmental aromatic pollutants (1Ramos J.L. Gonzalez-Perez M.M. Caballero A. van Dillewijn P. Curr. Opin. Biotechnol. 2005; 16: 275-281Crossref PubMed Scopus (83) Google Scholar, 2Diaz E. Int. Microbiol. 2004; 7: 173-180PubMed Google Scholar, 3Parales R.E. Haddock J.D. Curr. Opin. Biotechnol. 2004; 15: 374-379Crossref PubMed Scopus (81) Google Scholar, 4Komives T. Gullner G. Z. Naturforsch. 2005; 60: 179-185PubMed Google Scholar, 5D'Annibale A. Ricci M. Leonardi V. Quaratino D. Mincione E. Petruccioli M. Biotechnol. Bioeng. 2005; 90: 723-731Crossref PubMed Scopus (74) Google Scholar, 6Zhang C. Bennett G.N. Appl. Microbiol. Biotechnol. 2005; 67: 600-618Crossref PubMed Scopus (132) Google Scholar, 7Gibson J. Harwood C.S. Annu. Rev. Microbiol. 2002; 56: 345-369Crossref PubMed Scopus (179) Google Scholar, 8Samanta S.K. Singh O.V. Jain R.K. Trends Biotechnol. 2002; 20: 243-248Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar, 9Peres C.M. Agathos S.N. Biotechnol. Annu. Rev. 2000; 6: 197-220Crossref PubMed Scopus (69) Google Scholar, 10Chaudhry G.R. Chapalamadugu S. Microbiol. Rev. 1991; 55: 59-79Crossref PubMed Google Scholar, 11Haggblom M.M. FEMS Microbiol. Rev. 1992; 9: 29-71Crossref PubMed Google Scholar) and for chemical synthesis (3Parales R.E. Haddock J.D. Curr. Opin. Biotechnol. 2004; 15: 374-379Crossref PubMed Scopus (81) Google Scholar, 12Poppe L. Retey J. Angew. Chem. Int. Ed. Engl. 2005; 44: 3668-3688Crossref PubMed Scopus (105) Google Scholar, 13Boyd D.R. Sharma N.D. O'dowd C.R. Carroll J.G. Loke P.L. Allen C.C. Chem. Commun. 2005; 31: 3989-3991Crossref Scopus (18) Google Scholar, 14Boyd D.R. Sharma N.D. Llamas N.M. Malone J.F. O'Dowd C.R. Allen C.C. Org. Biomol. Chem. 2005; 3: 1953-1963Crossref PubMed Scopus (39) Google Scholar, 15Boyd D.R. Sharma N.D. Byrne B.E. Haughey S.A. Kennedy M.A. Allen C.C. Org. Biomol. Chem. 2004; 2: 2530-2537Crossref PubMed Scopus (40) Google Scholar). Studies of the aromatic pathways operative in common bacteria such as Escherichia coli and Pseudomonas putida (16Diaz E. Ferrandez A. Prieto M.A. Garcia J.L. Microbiol. Mol. Biol. Rev. 2001; 65: 523-569Crossref PubMed Scopus (289) Google Scholar, 17Jimenez J.L. Minambres B. Garcia L. Diaz E. Environ. Microbiol. 2002; 4: 824-841Crossref PubMed Scopus (385) Google Scholar) have provided a framework against which novel pathways of environmental bacteria can be understood (18Johnson G.R. Spain J.C. Appl. Microbiol. Biotechnol. 2003; 62: 110-123Crossref PubMed Scopus (61) Google Scholar, 19Bartolome-Martin D. Martinez-Garcia E. Mascaraque V. Rubio J. Perera J. Alonso S. Gene (Amst.). 2004; 341: 167-179Crossref PubMed Scopus (25) Google Scholar). In this paper, we focus on a pathway for aerobic phenylacetate degradation. Phenylacetate derives primarily from phenylalanine, but it is also formed in degradation pathways that target a variety of environmental aromatics of natural and synthetic origin and, thus, is the central element of an important catabolon (20Olivera E.R. Minambres B. Garcia B. Muniz C. Moreno M.A. Ferrandez A. Diaz E. Garcia J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (176) Google Scholar, 21Olivera E.R. Reglero A. Martinez-Blanco H. Fernandez-Medarde A. Moreno M.A. Luengo J.M. Eur. J. Biochem. 1994; 221: 375-381Crossref PubMed Scopus (31) Google Scholar, 22Luengo J.M. Garcia J.L. Olivera E.R. Mol. Microbiol. 2001; 39: 1434-1442Crossref PubMed Scopus (127) Google Scholar). The gene clusters that encode the phenylacetate pathway enzymes typically contain structural genes, regulatory genes, and transport genes (17Jimenez J.L. Minambres B. Garcia L. Diaz E. Environ. Microbiol. 2002; 4: 824-841Crossref PubMed Scopus (385) Google Scholar, 19Bartolome-Martin D. Martinez-Garcia E. Mascaraque V. Rubio J. Perera J. Alonso S. Gene (Amst.). 2004; 341: 167-179Crossref PubMed Scopus (25) Google Scholar, 20Olivera E.R. Minambres B. Garcia B. Muniz C. Moreno M.A. Ferrandez A. Diaz E. Garcia J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (176) Google Scholar, 22Luengo J.M. Garcia J.L. Olivera E.R. Mol. Microbiol. 2001; 39: 1434-1442Crossref PubMed Scopus (127) Google Scholar, 23Ferrandez A. Minambres B. Garcia B. Olivera E.R. Luengo J.M. Garcia J.L. Diaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 24Mohamed M.E.-S. Ismail W. Heider J. Fuchs G. Arch. Microbiol. 2002; 178: 180-192Crossref PubMed Scopus (68) Google Scholar, 25Rost R. Haas S. Hammer E. Herrmann H. Burchhardt G. Mol. Genet. Genomics. 2002; 267: 656-663Crossref PubMed Scopus (39) Google Scholar, 26Gescher J. Zaar A. Mohamed M. Schagger H. Fuchs G. J. Bacteriol. 2002; 184: 6301-6315Crossref PubMed Scopus (92) Google Scholar, 27Alonso S. Bartolome-Martin D. del Alamo M. Diaz E. Garcia J.L. Perera J. Gene (Amst.). 2003; 319: 71-83Crossref PubMed Scopus (23) Google Scholar). In E. coli, the 14 open reading frame cluster includes the structural genes paaABCDEFGHIJKXYZ (23Ferrandez A. Minambres B. Garcia B. Olivera E.R. Luengo J.M. Garcia J.L. Diaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Only one of these genes, paaK, has been assigned function through isolation and kinetic characterization of its protein product, phenylacetate-CoA ligase (20Olivera E.R. Minambres B. Garcia B. Muniz C. Moreno M.A. Ferrandez A. Diaz E. Garcia J.L. Luengo J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6419-6424Crossref PubMed Scopus (176) Google Scholar, 23Ferrandez A. Minambres B. Garcia B. Olivera E.R. Luengo J.M. Garcia J.L. Diaz E. J. Biol. Chem. 1998; 273: 25974-25986Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 28Martinez-Blanco H. Reglero A. Rodriguez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar, 29Mohamed M.E.-S. J. Bacteriol. 2000; 182: 286-294Crossref PubMed Scopus (44) Google Scholar). This ligase catalyzes the first committed step of the pathway, which is the conversion of the ring acetate group to the corresponding CoA thioester (28Martinez-Blanco H. Reglero A. Rodriguez-Aparicio L.B. Luengo J.M. J. Biol. Chem. 1990; 265: 7084-7090Abstract Full Text PDF PubMed Google Scholar, 29Mohamed M.E.-S. J. Bacteriol. 2000; 182: 286-294Crossref PubMed Scopus (44) Google Scholar) (Fig. 1). Phenylacetyl-CoA then induces the synthesis of the other pathway enzymes (30Ferrandez A. Garcia J.L. Diaz E. J. Biol. Chem. 2000; 275: 12214-12225Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 31Garcia B. Olivera E.R. Minambres B. Carnicero D. Muniz C. Naharro G. Luengo J.M. Appl. Environ. Microbiol. 2000; 66: 4575-4578Crossref PubMed Scopus (27) Google Scholar). The reactions catalyzed by these enzymes have been investigated but not firmly demonstrated (32Ismail W. Mohamed M.E.-S. Wanner B.L. Datsenko K.A. Eisenreich W. Rohdich F. Bacher A. Fuchs G. Eur. J. Biochem. 2003; 270: 3047-3054Crossref PubMed Scopus (66) Google Scholar). Nevertheless, a working model that incorporates the findings from the studies of both the phenylacetate pathway (32Ismail W. Mohamed M.E.-S. Wanner B.L. Datsenko K.A. Eisenreich W. Rohdich F. Bacher A. Fuchs G. Eur. J. Biochem. 2003; 270: 3047-3054Crossref PubMed Scopus (66) Google Scholar) and the analogous benzoate pathway of Azoarcus evansii (26Gescher J. Zaar A. Mohamed M. Schagger H. Fuchs G. J. Bacteriol. 2002; 184: 6301-6315Crossref PubMed Scopus (92) Google Scholar, 33Gescher J. Eisenreich W. Worth J. Bacher A. Fuchs G. Mol. Microbiol. 2005; 56: 1586-1600Crossref PubMed Scopus (43) Google Scholar, 34Zaar A. Gescher J. Eisenreich W. Bacher A. Fuchs G. Mol. Microbiol. 2004; 54: 223-238Crossref PubMed Scopus (40) Google Scholar) can be inferred (Fig. 1). According to this model, the PaaABCD enzyme complex catalyzes the addition of oxygen across the ring C(1)=C(2) bond of phenylacetyl-CoA followed by reduction to the dihydrodiol (32Ismail W. Mohamed M.E.-S. Wanner B.L. Datsenko K.A. Eisenreich W. Rohdich F. Bacher A. Fuchs G. Eur. J. Biochem. 2003; 270: 3047-3054Crossref PubMed Scopus (66) Google Scholar). The next hypothetical step is ring opening to the C-8 aldehyde, which is converted to the corresponding acid. The ensuing CoA thiolytic cleavage forms a C-6 CoA ester (32Ismail W. Mohamed M.E.-S. Wanner B.L. Datsenko K.A. Eisenreich W. Rohdich F. Bacher A. Fuchs G. Eur. J. Biochem. 2003; 270: 3047-3054Crossref PubMed Scopus (66) Google Scholar), which is processed via β-oxidation by enzymes of the crotonase superfamily (35Holden H.M. Benning M.M. Haller T. Gerlt J.A. Acc. Chem. Res. 2001; 34: 145-157Crossref PubMed Scopus (162) Google Scholar, 36Haller T. Buckel T. Retey J. Gerlt J.A. Biochemistry. 2000; 39: 4622-4629Crossref PubMed Scopus (125) Google Scholar). The one gene of the pathway gene cluster, paaABCDEFGHIJKXYZ, that does not have a defined role in phenylacetate degradation is paaI. The protein product PaaI is a member of the hot dog-fold enzyme superfamily (37Leesong M. Henderson B.S. Gillig J.R. Schwab J.M. Smith J.L. Structure. 1996; 4: 253-264Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 38Dillon S.C. Bateman A. BMC Bioinformatics. 2004; 5: 109-122Crossref PubMed Scopus (146) Google Scholar). Moreover, it belongs to the acyl-CoA thioesterase subfamily and, in particular, to the same clade of acyl-CoA thioesterases as does the Arthrobacter 4-hydroxybenzoyl-coenzyme A thioesterase (4-HBA-CoA thioesterase) 3The abbreviations used are: 4-HBA, 4-hydoxybenzoate; CoA, coenzyme A; DTT, dithio-threitol; ESI, electrospray ionization; HPLC, high performance liquid chromatography; PDB, Protein Data Bank; WT, wild type. (39Zhuang Z. Gartemann K.-H. Eichenlaub R. Dunaway-Mariano D. Appl. Environ. Microbiol. 2003; 69: 2707-2711Crossref PubMed Scopus (30) Google Scholar, 40Thoden J.B. Zhuang Z. Dunaway-Mariano D. Holden H.M. J. Biol. Chem. 2003; 278: 43709-43716Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Acyl-CoA thioesterases generally function in the cell to release the carboxylate unit for degradation, export (39Zhuang Z. Gartemann K.-H. Eichenlaub R. Dunaway-Mariano D. Appl. Environ. Microbiol. 2003; 69: 2707-2711Crossref PubMed Scopus (30) Google Scholar, 41Scholten J.D. Chang K.-H. Babbitt P.C. Charest H. Sylvestre M. Dunaway-Mariano D. Science. 1991; 253: 182-185Crossref PubMed Scopus (129) Google Scholar, 42Zhuang Z. Song F. Takami H. Dunaway-Mariano D. J. Bacteriol. 2004; 186: 393-399Crossref PubMed Scopus (17) Google Scholar, 43Hunt M.C. Alexson S.E. Prog. Lipid Res. 2002; 41: 99-130Crossref PubMed Scopus (220) Google Scholar, 44Yu W. Liang X. Ensenauer R.E. Vockley J. Sweetman L. Schulz H. J. Biol. Chem. 2004; 279: 52160-52167Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 45Ren Y. Aguirre J. Ntamack A.G. Chu C. Schulz H. J. Biol. Chem. 2004; 279: 11042-11050Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), or regulation (43Hunt M.C. Alexson S.E. Prog. Lipid Res. 2002; 41: 99-130Crossref PubMed Scopus (220) Google Scholar, 46Duncan J.A. Gilman A.G. J. Biol. Chem. 1998; 273: 15830-15837Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 47Mumby S.M. Kleuss C. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2800-2804Crossref PubMed Scopus (222) Google Scholar, 48Hunt M.C. Solaas K. Kase B.F. Alexson S.E. J. Biol. Chem. 2002; 277: 1128-1138Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and/or to release the CoA in thioester metabolites for participation in alternate metabolic pathways (26Gescher J. Zaar A. Mohamed M. Schagger H. Fuchs G. J. Bacteriol. 2002; 184: 6301-6315Crossref PubMed Scopus (92) Google Scholar). In this paper we report and interpret the results from substrate screens of A. evansii and E. coli PaaI to define the biochemical role of PaaI in the context of the phenylacetate pathway. The x-ray structure of the E. coli apo PaaI is also reported. The E. coli PaaI and Thermus thermophilus PaaI (49Kunishima N. Asada Y. Sugahara M. Ishijima J. Nodake Y. Sugahara M. Miyano M. Kuramitsu S. Yokoyama S. Sugahara M. J. Mol. Biol. 2005; 352: 212-228Crossref PubMed Scopus (48) Google Scholar) structures are analyzed in the context of the PaaI substrate specificity profile and the kinetic properties of the PaaI active site mutants. A mechanism for PaaI substrate recognition and catalysis is proposed. Chemicals—All restriction enzymes and the T4 DNA ligase were purchased from Invitrogen. Pfu Turbo DNA polymerase was purchased from Stratagene. Oligonucleotide primers were custom-synthesized by Invitrogen. DNA sequencing was performed by the DNA Sequencing Facility of the University of New Mexico. Acetyl-CoA, crotonyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isobutyryl-CoA, β-hydroxybutyryl-CoA, n-hexanoyl-CoA, and phenylacetyl-CoA were purchased from Sigma. 4-Hydroxybenzoyl-CoA, 3-hydroxybenzoyl-CoA, 4-hydroxyphenacyl-CoA, 3-hydroxyphenacyl-CoA, 4-hydroxybenzyl-CoA, 4-chlorobenzoyl-CoA, 4-methoxybenzoyl-CoA, 4-hydroxyphenylacetyl-CoA, 3-hydroxyphenylacetyl-CoA, 3,4-dihydroxyphenylacetyl-CoA, and 3,5-dihydroxyphenylacetyl-CoA were synthesized as reported (50Thoden J.B. Holden H.M. Zhuang Z. Dunaway-Mariano D. J. Biol. Chem. 2002; 277: 27468-27476Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 51Luo L. Taylor K.L. Xiang H. Wei Y. Zhang W. Dunaway-Mariano D. Biochemistry. 2001; 40: 15684-15692Crossref PubMed Scopus (37) Google Scholar, 52Merkel S.M. Eberhard A.E. Gibson J. Harwood C.S. J. Bacteriol. 1989; 171: 1-7Crossref PubMed Google Scholar). All the starting materials for these syntheses were purchased from Sigma except for 3,5-dihydroxyphenylacetate, which was synthesized from diethyl 1,3-acetone dicarboxylate according to Theilacker and Schmid (53Theilacker W. Schmid W. Annalen. 1950; 570: 15-33Crossref Scopus (17) Google Scholar) as detailed in the supplemental material. Crystallographic Analysis—C-Terminal His-tagged E. coli PaaI was prepared for crystallization as detailed in the supplemental material. The N-terminal Met was replaced by Ser-Leu. Crystals of the selenomethionine (54Hendrickson W.A. Horton J.R. LeMaster D.M. EMBO J. 1990; 9: 1665-1672Crossref PubMed Scopus (1008) Google Scholar) PaaI were grown in 13% polyethylene glycol (average molecularweight,4000),0.1m[bis(2-hydroxyethyl)imino]-tris(hydroxymethyl)methane (pH 6.0) and 0.2 m lithium sulfate at 18 °C using the hanging drop vapor diffusion method. They were incubated in the presence of 30% glycerol before being flash-frozen in liquid nitrogen. The crystals belong to trigonal space group P3121 with a = b = 69.9 Å and c = 117.2 Å. X-ray diffraction data were collected at APS beamline 31ID at wavelength 0.9798 Å. Data were reduced with DENZO, SCALEPACK (55Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38609) Google Scholar), and CCP4 (56Number Collaborative Computational Project Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). The structure of PaaI was determined using SAD technique employing the anomalous signal from selenium atoms as implemented in the software SOLVE (57Terwilliger T.C. Berendzen J. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar). Ten selenium atoms were located, and phases derived from these anomalous centers were used for density modification as implemented in the software RESOLVE (58Terwilliger T.C. Acta Crystallogr. D Biol. Crystallogr. 2000; 56: 965-972Crossref PubMed Scopus (1635) Google Scholar). Manual model building was accomplished using O (59Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-118Crossref PubMed Scopus (13014) Google Scholar). Refinement of the model was accomplished using CNS (60Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar) and Refmac as implemented in CCP4 (56Number Collaborative Computational Project Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar). Water molecules were located and refined. The final model contains 2016 protein atoms, 79 solvent atoms, and 15 sulfate atoms and was refined to an R value of 18.7% and an Rfree value of 23.0%. Crystallographic details are in Table 3.TABLE 3Crystallographic data and refinement statisticsCell constantsa = b = 69.9Å, c = 117.2ÅSpace groupP3121, 2 molecules per asymmetric unitX-ray sourceAPS 31ID BeamlinePDB code2FS2Se-Met peak dataWavelength (Å)0.9798Resolution (Å)20.0-2.0Number of observations99,872Number of reflectionsaData completeness treats Bijvoët mates independently40,460Completeness (%) (2.07–2.0 Å)aData completeness treats Bijvoët mates independently93.5 (88.9)Average redundancy (2.07–2.0 Å)aData completeness treats Bijvoët mates independently2.5 (2.2)Mean I/σ(I) (2.07-2.0 Å)6.9 (1.4)Rmerge on IbRmerge = ΣhklΣi|I(hkl)i – <I(hkl)>|/ΣhklΣi <I(hkl)i> (2.07-2.0 Å)9.3 (46.2)Cut-off criteriaI > –0.5 σ(I)SOLVE figure of meritcFigure of merit was calculated using SOLVE/RESOLVE0.21 (20.0-2.0 Å of resolution) for 19,074 reflectionsRESOLVE Figure of meritc with 2-fold NCS0.48 (20.0-2.6 Å of resolution) for 29,562 reflectionsModel and refinement statisticsResolution range20.0-2.0 ÅNumber of reflections19,072 (18,102 in working set; 970 in test set)Completeness83.1% (5.1% in test set)Cutoff criterionF > 0.0Protein atoms2016Water atoms79Sulfate atoms15RcrystdRcryst = Σhkl|Fo (hkl) – Fc (hkl)|/Σhkli|Fo (hkl)|, where Fo and Fc are observed and calculated structure factors, respectively (2.05–2.0 Å)0.187 (0.319)Rfree (2.05–2.0 Å)0.230 (0.366)Root mean square deviationsBond lengths (Å)0.031Bond angles (Å)2.3Mean B value/B factor root mean square deviation main chain/side chain (Å2)32.9/1.7/4.8Ramachandran plot statisticseCalculated with PROCHECKResidues in most favored regions224 (93.3%)Residues in additional allowed regions16 (6.7%)Residues in generously allowed regions0 (0%)Residues in disallowed regions0 (0%)a Data completeness treats Bijvoët mates independentlyb Rmerge = ΣhklΣi|I(hkl)i – <I(hkl)>|/ΣhklΣi <I(hkl)i>c Figure of merit was calculated using SOLVE/RESOLVEd Rcryst = Σhkl|Fo (hkl) – Fc (hkl)|/Σhkli|Fo (hkl)|, where Fo and Fc are observed and calculated structure factors, respectivelye Calculated with PROCHECK Open table in a new tab Preparation of Wild-type and Mutant A. evansii and E. coli PaaI for Kinetic Study—A. evansii (accession number Q9F9VO) and E. coli paaI (accession number P76084) genes were amplified by PCR (61Erlich H.A. PCR Technology: Principles and Applications for DNA Amplification. W. H. Freeman and Co., New York, NY1991Google Scholar) using genomic DNA prepared from A. evansii DSM 6898 (DSMZ) and E. coli strain ATCC 11105 (Manassas, VA) as templates, respectively, commercial oligonucleotides as primers, and Pfu Turbo as the DNA polymerase. The PCR products were digested with the restriction enzymes NdeI and HindIII and then purified by agarose gel chromatography before T4 DNA ligase catalyzed the ligation to pET-23b (+) vector (Novagen) digested by the NdeI and HindIII. The resulting clones named EcWT-PaaI/pET-23b and AeWT-PaaI/pET-23b were verified by DNA sequencing. Mutagenesis was carried out using a PCR-based strategy with commercial primers and the WT-PaaI/pET-23b (+) plasmid serving as template. The purified PCR products were used to transform to E. coli JM109-competent cells (Stratagene). Mutant genes were verified by DNA sequencing. For protein production PaaI/pET-23b transformants of E. coli BL21(DE3) were grown aerobically at 28 °C in LB media containing 50 μg/ml carbenicillin for ∼10 h (A600 1.0) then induced with 0.4 mm isopropyl-β-d-galactopyranoside for 5 h. Cells were harvested by centrifugation (5000 × g for 10 min), suspended in 100 ml of 50 mm K+Hepes, 0.1 mm phenylmethylsulfonyl fluoride, 1 mm DTT (pH 7.5, 0 °C) and passed through a French press at 1200 p.s.i. twice before centrifugation at 48,000 × g for 30 min at 4 °C. The supernatant was applied to a 45 × 3.5-cm DEAE-Sepharose (Amersham Biosciences) column and eluted at 4 °C with a 2-liter gradient of 0-0.5 m KCl in 50 mm K+Hepes, 1 mm DTT (pH 7.5). The thioesterase-containing fractions (eluted at ∼ 0.3 m KCl) were combined and then precipitated with ammonium sulfate (40-60% cut for A. evansii and 0-40% cut for E. coli PaaI). The A. evansii PaaI was dissolved in 2 ml of 10 mm K+Hepes (pH 7.5) buffer with 0.15 m KCl and 1 mm DTT, and E. coli PaaI was dissolved in 2 ml of 10 mm Tris (pH 8.1) buffer with 0.05 m KCl and 1 mm DTT. The protein solution (1 ml) was applied to a 100 × 2-cm Sephacryl S-200 column (Amersham Biosciences) and eluted at 4 °C with the same buffer at 0.5 ml/min. The thioesterase-containing fractions were pooled and concentrated with a 10-kDa Macrosep centricon (Pall Filtron). The activity of the A. evansii PaaI was stable for storage in 10 mm K+Hepes (pH 7.5) buffer or in 10 mm Tris (pH 8.1) containing 10 mm KCl at -80 °C for several months. The E. coli PaaI could be stored in 10 mm Tris (pH 8.1) at 0 °C (not frozen) ice for several weeks without significant loss of activity. The yields of homogeneous (based on SDS-PAGE analysis) A. evansii PaaI proteins in mg/g of wet cells were WT (11Haggblom M.M. FEMS Microbiol. Rev. 1992; 9: 29-71Crossref PubMed Google Scholar), D75A (11Haggblom M.M. FEMS Microbiol. Rev. 1992; 9: 29-71Crossref PubMed Google Scholar), D75N (9Peres C.M. Agathos S.N. Biotechnol. Annu. Rev. 2000; 6: 197-220Crossref PubMed Scopus (69) Google Scholar), N60A (9Peres C.M. Agathos S.N. Biotechnol. Annu. Rev. 2000; 6: 197-220Crossref PubMed Scopus (69) Google Scholar) and N60D (7Gibson J. Harwood C.S. Annu. Rev. Microbiol. 2002; 56: 345-369Crossref PubMed Scopus (179) Google Scholar), whereas those for the E. coli PaaI proteins were WT (50Thoden J.B. Holden H.M. Zhuang Z. Dunaway-Mariano D. J. Biol. Chem. 2002; 277: 27468-27476Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), D61A (4Komives T. Gullner G. Z. Naturforsch. 2005; 60: 179-185PubMed Google Scholar), N46A (9Peres C.M. Agathos S.N. Biotechnol. Annu. Rev. 2000; 6: 197-220Crossref PubMed Scopus (69) Google Scholar), E14A (22Luengo J.M. Garcia J.L. Olivera E.R. Mol. Microbiol. 2001; 39: 1434-1442Crossref PubMed Scopus (127) Google Scholar), N15A (10Chaudhry G.R. Chapalamadugu S. Microbiol. Rev. 1991; 55: 59-79Crossref PubMed Google Scholar), D16A (7Gibson J. Harwood C.S. Annu. Rev. Microbiol. 2002; 56: 345-369Crossref PubMed Scopus (179) Google Scholar), H52A (6Zhang C. Bennett G.N. Appl. Microbiol. Biotechnol. 2005; 67: 600-618Crossref PubMed Scopus (132) Google Scholar), and N46Q/D61E (8Samanta S.K. Singh O.V. Jain R.K. Trends Biotechnol. 2002; 20: 243-248Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar). Fast protein liquid chromatography gel filtration chromatography (Hiload 60 × 16-cm Amersham Biosciences Superdex S-200) was used to determine whether the A. evansii PaaI mutant proteins were contaminated with E. coli PaaI. A solution of 10 mm K+Hepes (pH 7.5) containing 0.15 m KCl and 1 mm DTT was used to elute the column at a flow rate of 1 ml/min. The retention time of the E. coli PaaI was 52.3 min versus 47.0 min for the A. evansii PaaI. The activities of the A. evansii PaaI mutants were measured before and after chromatography and found to be identical, thereby demonstrating that the protein purification protocol described above removes E. coli PaaI from the A. evansii PaaI. Molecular Size Determination—The molecular mass was calculated from the amino acid composition, derived from the gene sequence, by using the EXPASY Molecular Biology Server program Compute pI/Mw. The molecular mass was measured by ESI mass spectrometry and by SDS-PAGE (4% stacking gel and 18% separating gel). The mass of native PaaI was estimated by fast protein liquid chromatography gel filtration column chromatography carried out as described in the previous section. Commercial protein molecular weight standards (Amersham Biosciences) were used to generate a plot of log Mr versus elution volume from the column. Kinetic Assays—Steady-state kinetic methods were used to determine the kcat and Km for wild-type and mutant PaaI as a function of substrate screening, reaction solution pH, or presence of an inhibitor. The details of each experimental measurement are provided in the supplemental material. The A. evansii PaaI reactions were typically monitored at 25 °C by using a 5,5′-dithio-bis(2-nitrobenzoic acid)-based assay in which the absorbance of 5-thio-2-nitrobenzoate at 412 nm (Δϵ = 13.6 mm-1·cm-1) was measured. For E. coli PaaI, which is rapidly inactivated by 5,5′-dithio-bis(2-nitrobenzoic acid under assay conditions, a continuous spectrophotometric assay was employed. Accordingly, the decrease in solution absorbance at 236 nm was monitored for the reaction mixtures of 4-hydroxyphenylacetyl-CoA (Δϵ = 4.2 mm-1·cm-1), 3-hydroxyphenylacetyl-CoA (Δϵ = 3.6 mm-1·cm-1), and 3,4-dihydroxyphenylacetyl-CoA (Δϵ = 3.2 mm-1·cm-1). For the phenylacetyl-CoA substrates, a fixed-time, reversed-phase HPLC-based assay was used. The initial velocity data, measured as a function of substrate concentration, were analyzed using Equation 1 V=Vmax[S]/([S]+Km)(Eq. 1) where V is initial velocity, Vmax is maximum velocity, [S] is substrate concentration, and Km is the Michaelis constant. The kcat was calculated from Vmax/[E], where [E] is the total enzyme concentration (determined using the Bradford method (62Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217487) Google Scholar)). The inhibition constant Ki was obtained by fitting the initial rates to Equation 2 V=Vmax[S]/[Km(1+[I]/Ki)+[S]](Eq. 2) where [I] is the concentration of the inhibitor, and Ki is the inhibition constant. The log kcat and log(kcat/Km) values obtained from the pH rate profile analysis were fitted using Equation 3 logY=log(C/(1+[H]/Ka+Kb/[H]))(Eq. 3) where Y is kcat or kcat/Km,[H] is the hydrogen ion concentration, C is the pH independent value of kcat or kcat/Km, Ka is the acid dissociation constant, and Kb is the base dissociation constant. Data analysis was carried out using the computer program KinetAsyst (IntelliKinetics). The reported error was computed for the data fitting. Recombinant A. evansii and E. coli PaaI Characterization—The theoretical molecular weights of the A. evansii (154 amino acids) and E. coli (140 amino acids) PaaIs are 16,550 and 14,851, respectively, which compare with the ESI mass spectrometric determined molecular weights of 16,419 and 14,720. Both recombinant proteins have, thus, lost the N-terminal Met (-131 Da) by post-translational modification. The A. evansii and E. coli PaaIs migrate on SDS-PAGE gels as 15- and 14-kDa proteins, whereas the native molecular masses determined by gel filtration chromatography correspond to 67 and 58 kDa. Both PaaIs are, therefore, homotetramers. The pH rate profiles of PaaI-catalyzed 3,4-dihydroxyphenylacetyl-CoA hydrolysis (identified as the most active substrate; see below) were measured using initial-velocity techniques (Fig. 2). Both PaaIs are stable at pH 6-10 (Fig. 2, insets) and most active at pH 6-9. The bell-shaped curves were fitted to define the apparent pKa values for ionization of essential residues in the enzyme-substrate complex (log(kcat)) and in the uncomplexed enzyme and substrate" @default.
• W2144304328 created "2016-06-24" @default.
• W2144304328 creator A5013774625 @default.
• W2144304328 creator A5015599531 @default.
• W2144304328 creator A5043323040 @default.
• W2144304328 creator A5048900539 @default.
• W2144304328 creator A5050629095 @default.
• W2144304328 creator A5067739445 @default.
• W2144304328 creator A5070991573 @default.
• W2144304328 creator A5078818863 @default.
• W2144304328 creator A5090254750 @default.
• W2144304328 date "2006-04-01" @default.
• W2144304328 modified "2023-09-27" @default.
• W2144304328 title "Structure, Function, and Mechanism of the Phenylacetate Pathway Hot Dog-fold Thioesterase PaaI" @default.
• W2144304328 cites W1506811553 @default.
• W2144304328 cites W1510205119 @default.
• W2144304328 cites W1539796472 @default.
• W2144304328 cites W1561123533 @default.
• W2144304328 cites W1595057496 @default.
• W2144304328 cites W1676494442 @default.
• W2144304328 cites W1797216021 @default.
• W2144304328 cites W1855537617 @default.
• W2144304328 cites W1878988281 @default.
• W2144304328 cites W1887891908 @default.
• W2144304328 cites W1965277349 @default.
• W2144304328 cites W1967316786 @default.
• W2144304328 cites W1970196085 @default.
• W2144304328 cites W1974518253 @default.
• W2144304328 cites W1977017222 @default.
• W2144304328 cites W1977577081 @default.
• W2144304328 cites W1977649184 @default.
• W2144304328 cites W1980737963 @default.
• W2144304328 cites W1986103520 @default.
• W2144304328 cites W1990104653 @default.
• W2144304328 cites W1995017064 @default.
• W2144304328 cites W1998679041 @default.
• W2144304328 cites W1999132254 @default.
• W2144304328 cites W2001641653 @default.
• W2144304328 cites W2002403955 @default.
• W2144304328 cites W2004829400 @default.
• W2144304328 cites W2008867070 @default.
• W2144304328 cites W2013083986 @default.
• W2144304328 cites W2015475839 @default.
• W2144304328 cites W2016048379 @default.
• W2144304328 cites W2020848561 @default.
• W2144304328 cites W2022058405 @default.
• W2144304328 cites W2038988226 @default.
• W2144304328 cites W2046036240 @default.
• W2144304328 cites W2047316348 @default.
• W2144304328 cites W2050972936 @default.
• W2144304328 cites W2061361921 @default.
• W2144304328 cites W2064313065 @default.
• W2144304328 cites W2066438497 @default.
• W2144304328 cites W2073170899 @default.
• W2144304328 cites W2076533036 @default.
• W2144304328 cites W2079236526 @default.
• W2144304328 cites W2091308266 @default.
• W2144304328 cites W2093626204 @default.
• W2144304328 cites W2101295502 @default.
• W2144304328 cites W2103695796 @default.
• W2144304328 cites W2107214348 @default.
• W2144304328 cites W2116747675 @default.
• W2144304328 cites W2122044132 @default.
• W2144304328 cites W2123580563 @default.
• W2144304328 cites W2125873032 @default.
• W2144304328 cites W2126521605 @default.
• W2144304328 cites W2130970547 @default.
• W2144304328 cites W2132169354 @default.
• W2144304328 cites W2136984336 @default.
• W2144304328 cites W2140434332 @default.
• W2144304328 cites W2143694829 @default.
• W2144304328 cites W2151091061 @default.
• W2144304328 cites W2156243227 @default.
• W2144304328 cites W2158021691 @default.
• W2144304328 cites W2162884305 @default.
• W2144304328 cites W2170816545 @default.
• W2144304328 cites W4293247451 @default.
• W2144304328 doi "https://doi.org/10.1074/jbc.m513896200" @default.
• W2144304328 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16464851" @default.
• W2144304328 hasPublicationYear "2006" @default.
• W2144304328 type Work @default.
• W2144304328 sameAs 2144304328 @default.
• W2144304328 citedByCount "59" @default.
• W2144304328 countsByYear W21443043282012 @default.
• W2144304328 countsByYear W21443043282013 @default.
• W2144304328 countsByYear W21443043282014 @default.
• W2144304328 countsByYear W21443043282015 @default.
• W2144304328 countsByYear W21443043282016 @default.
• W2144304328 countsByYear W21443043282017 @default.
• W2144304328 countsByYear W21443043282018 @default.
• W2144304328 countsByYear W21443043282019 @default.
• W2144304328 countsByYear W21443043282020 @default.
• W2144304328 countsByYear W21443043282022 @default.
• W2144304328 countsByYear W21443043282023 @default.
• W2144304328 crossrefType "journal-article" @default.
• W2144304328 hasAuthorship W2144304328A5013774625 @default.
• W2144304328 hasAuthorship W2144304328A5015599531 @default.
• W2144304328 hasAuthorship W2144304328A5043323040 @default.

• next