Site-directed Mutagenesis of 2,4-Dichlorophenoxyacetic Acid/α-Ketoglutarate Dioxygenase
2000; Elsevier BV; Volume: 275; Issue: 17 Linguagem: Inglês
10.1074/jbc.275.17.12400
ISSN1083-351X
AutoresDeborah A. Hogan, Sheila R. Smith, Eric A. Saari, John McCracken, Robert P. Hausinger,
Tópico(s)Cancer, Hypoxia, and Metabolism
Resumo2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the α-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX 50–70HX 10RXS arrangement of residues that make up the binding sites for Fe(II) and α-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X 138–207 HX 10R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5α; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K m values for 2,4-D, and the former showed a decreased k cat, suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D. 2,4-Dichlorophenoxyacetic acid (2,4-D)/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. The active site structures of a small number of enzymes within the α-KG-dependent dioxygenase superfamily have been characterized and shown to have a similar HXDX 50–70HX 10RXS arrangement of residues that make up the binding sites for Fe(II) and α-KG. TfdA does not have obvious homology to the dioxygenases containing the above motif but is related in sequence to eight other enzymes in the superfamily that form a distinct consensus sequence (HX(D/E)X 138–207 HX 10R/K). Variants of TfdA were created to examine the roles of putative metal-binding residues and the functions of the other seven histidines in this protein. The H167A, H200A, H213A, H245A, and H262A forms of TfdA formed inclusion bodies when overproduced in Escherichia coli DH5α; however, these proteins were soluble when fused to the maltose-binding protein (MBP). MBP-TfdA exhibited kinetic parameters similar to the native enzyme. The H8A and H235A variants were catalytically similar to wild-type TfdA. MBP-H213A and H216A TfdA have elevated K m values for 2,4-D, and the former showed a decreased k cat, suggesting these residues may affect substrate binding or catalysis. The H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A and MBP-H262A variants of TfdA were inactive. Gel filtration analysis revealed that the latter two proteins were highly aggregated. The remaining four inactive variants were examined in their Cu(II)-substituted forms by EPR and electron spin-echo envelope modulation (ESEEM) spectroscopic methods. Changes in EPR spectra upon addition of substrates indicated that copper was present at the active site in the H113A and D115A variants. ESEEM analysis revealed that two histidines are bound equatorially to the copper in the D115A and MBP-H167A TfdA variants. The experimental data and sequence analysis lead us to conclude that His-113, Asp-115, and His-262 are likely metal ligands in TfdA and that His-213 may aid in catalysis or binding of 2,4-D. 4-D, 2,4-dichlorophenoxyacetic acid α-ketoglutarate 2,4-D/α-KG dioxygenase taurine/α-KG dioxygenase continuous wave electron paramagnetic resonance electron spin echo envelope modulation isopenicillin N synthase deacetoxycephalosporin synthase maltose-binding protein Fourier transform 4-morpholinepropanesulfonic acid 2,4-Dichlorophenoxyacetic acid (2,4-D)1/α-ketoglutarate (α-KG) dioxygenase (TfdA) is an Fe(II)- and α-KG-dependent enzyme that catalyzes the first step in degradation of the herbicide 2,4-D. This enzyme couples the oxidative decarboxylation of α-KG to the hydroxylation of a side chain carbon atom. The resultant hemiacetal spontaneously decomposes to form 2,4-dichlorophenol and glyoxalate (1.Fukumori F. Hausinger R.P. J. Bacteriol. 1993; 175: 2083-2086Crossref PubMed Google Scholar). Mechanistically, TfdA resembles numerous other α-KG-dependent dioxygenases from plants, animals, fungi, and bacteria that catalyze similar hydroxylation reactions at unactivated carbon centers (2.De Carolis E. De Luca V. Phytochemistry. 1994; 36: 1093-1107Crossref PubMed Scopus (95) Google Scholar, 3.Prescott A.G. John P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 245-271Crossref PubMed Scopus (232) Google Scholar). Members of the α-KG-dependent dioxygenase superfamily are not closely related by their sequences but rather appear to fall into one of three groups of related enzymes or fall into a fourth group of unrelated sequences (4.Hogan D.A. Characterization and Function of 2,4-Dichlorophenoxyacetic Acid/α-Ketoglutarate Dioxygenase and Related Enzymes Ph.D. thesis. Michigan State University, East Lansing, MI1999Google Scholar). The best studied α-KG-dependent hydroxylases, including prolyl and lysyl hydroxylase (5.Kivirikko K.I. Pihlajaniemi T. Adv. Enzymol. Relat. Areas Mol. Biol. 1998; 72: 328-398Google Scholar) and flavanone hydroxylase (3.Prescott A.G. John P. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 245-271Crossref PubMed Scopus (232) Google Scholar, 6.Hedden P. Biochem. Soc. Trans. 1992; 20: 373-377Crossref PubMed Scopus (20) Google Scholar), have an HXDX∼55HX 10RXS motif in common (7.Borovok I. Landman O. Kreisberg-Zakarin R. Aharonowitz Y. Cohen G. Biochemistry. 1996; 35: 1981-1987Crossref PubMed Scopus (68) Google Scholar, 8.Lukacin R. Britsch L. Eur. J. Biochem. 1997; 249: 748-757Crossref PubMed Scopus (122) Google Scholar), and this motif is present in the more than 20 enzymes, defined here as Group I, within the α-KG dependent dioxygenase superfamily (8.Lukacin R. Britsch L. Eur. J. Biochem. 1997; 249: 748-757Crossref PubMed Scopus (122) Google Scholar, 9.Kreisberg-Zakarin R. Borovok I. Yanko M. Aharonowitz Y. Cohen G. Antonie Van Leeuwenhoek. 1999; 75: 33-39Crossref PubMed Scopus (17) Google Scholar). Site-directed mutagenesis studies have confirmed the importance of these residues for activity (8.Lukacin R. Britsch L. Eur. J. Biochem. 1997; 249: 748-757Crossref PubMed Scopus (122) Google Scholar, 10.Myllyharju J. Kivirikko K.I. EMBO J. 1997; 16: 1173-1180Crossref PubMed Scopus (164) Google Scholar, 11.Lamberg A. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1995; 270: 9926-9931Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 12.Pirskanen A. Kaimio A.-M. Myllylä R. Kivirikko K.I. J. Biol. Chem. 1996; 271: 9398-9402Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 13.McGinnis K. Ku G.M. VanDusen W.J. Fu J. Garsky V. Stern A.M. Friedman P.A. Biochemistry. 1996; 35: 3957-3962Crossref PubMed Scopus (39) Google Scholar), and the crystal structures of two Group I enzymes, isopenicillinN synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), indicate that these residues comprise the metallocenter and α-KG-binding site (14.Lloyd M.D. Lee H.J. Harlos K. Zhang Z.H. Baldwin J.E. Schofield C.J. Charnock J.M. Garner C.D. Hara T. Terwisscha van Scheltinga A.C. Valegard K. Viklund J.A. Hajdu J. Andersson I. Danielsson A. Bhikhabhai R. J. Mol. Biol. 1999; 287: 943-960Crossref PubMed Scopus (105) Google Scholar, 15.Roach P.L. Clifton I.J. Fülöp V. Harlos K. Barton G.J. Hajdu J. Andersson I. Schofield C.J. Baldwin J.E. Nature. 1995; 375: 700-704Crossref PubMed Scopus (382) Google Scholar, 16.Roach P.L. Clifton I.J. Hensgens C.M.H. Shibata N. Schofield C.J. Hajdu J. Baldwin J.E. Nature. 1997; 387: 827-830Crossref PubMed Scopus (392) Google Scholar, 17.Valegård K. van Scheltinga A.C.T. Lloyd M.D. Hara T. Ramaswamy S. Perrakis A. Thompson A. Lee H.-J. Baldwin J.E. Schofield C.J. Hajdu J. Andersson I. Nature. 1998; 394: 805-809Crossref PubMed Scopus (315) Google Scholar). TfdA is not closely related in sequence to the Group I α-KG-dependent dioxygenases described above but is clearly homologous (25–30% identity) toEscherichia coli taurine/α-KG dioxygenase (TauD) (18.Eichhorn E. van der Ploeg J.R. Kertesz M.A. Leisinger T. J. Biol. Chem. 1997; 272: 23031-23036Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and sulfonate/α-KG dioxygenase from Saccharomyces cerevisiae(19.Hogan D.A. Auchtung T.A. Hausinger R.P. J. Bacteriol. 1999; 181: 5876-5879Crossref PubMed Google Scholar). Furthermore, PSI-BLAST analyses (20.Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) find additional relationships to γ-butyrobetaine hydroxylase and clavaminate synthase. Alignment of these Group II sequences indicates the conservation of two histidines and one aspartate (His-113, His-262, and Asp 115 in TfdA) as well as an invariant arginine that may be analogous to the α-KG-binding arginine in DAOCS and related enzymes (TableI). A third set of enzyme sequences (Group III) from members of the α-KG-dependent dioxygenase superfamily, including phytanoyl-CoA hydroxylase and proline hydroxylase, exhibit the presence of a third related motif despite the lack of overall sequence similarity to Group I or Group II enzymes.Table IConsensus motifs for subgroups within the α-KG-dependent dioxygenase superfamilyGroup I (IPNS, DAOCS, and related enzymes)HXDX 50–70HX 10(R/K)XSGroup II (TfdA, TauD, clavaminate synthase and related enzymes)HX(D/E)X 138–207HX 10–13RGroup III (Phytanoyl-CoA hydroxylase, proline hydroxylase and related enzymes)HXDX 72–101HX 10(R/K)XS Open table in a new tab In this study, we used site-directed mutagenesis methods to examine the roles of potential metal-binding residues in the above motif (His-113, His-262, and Asp-115) and the remaining seven histidines in TfdA. Previously published work showed that TfdA was inactivated by diethylpyrocarbonate, a histidine-selective reagent, and provided evidence consistent with the presence of multiple histidines in the active site (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar). Spectroscopic studies of TfdA showed the presence of two equatorially bound imidazole nitrogens as ligands to the active site metal and indicated that one imidazole ligand may be displaced or shifted to an axial position upon substrate binding (22.Cosper N.J. Stålhandske C.M.V. Saari R.E. Hausinger R.P. Scott R.A. J. Biol. Inorg. Chem. 1999; 4: 122-129Crossref PubMed Scopus (25) Google Scholar, 23.Whiting A.K. Que Jr., L. Saari R.E. Hausinger R.P. Fredrick M.A. McCracken J. J. Am. Chem. Soc. 1997; 119: 3413-3414Crossref Scopus (28) Google Scholar, 24.Hegg E.L. Whiting A.K. McCracken J. Hausinger R.P. Que J., L. Biochemistry. 1999; 38: 16714-16726Crossref PubMed Scopus (70) Google Scholar). Based on analyses of different TfdA variants, we identify several likely metal ligands and provide evidence that another one or two histidines may aid in substrate binding. All plasmids were constructed from pUS311 (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar), a pUC19 derivative that contains the Ralstonia eutropha JMP134 tfdA gene (Fig.1). The H8A, D115A, H213A, H216A, H235A, H245A, and H262A TfdA variants were created by direct mutation oftfdA in pUS311 by the Stratagene Quickchange System (Stratagene, La Jolla, CA). All mutagenic primers are listed in TableII. Two alternative approaches were used to construct the three remaining variants. Plasmids encoding H113A and H167A TfdA variants were created by CLONTECHmutagenesis of pXHtfdA, a pUC19 plasmid containing the 5′-XbaI-HindII fragment of the tfdAgene (Fig. 1). To create the complete gene containing the indicated mutations, the XbaI-HindII fragment was cloned into pHKtfdA, which contains the 3′ end of the tfdA gene. pHKtfdA was constructed in two steps. First, the 1.4-kilobase pairXbaI-SalI fragment from pUS311, containing the complete tfdA gene, was cloned into pBC KS−(Stratagene) cut with XbaI and XhoI to create pBCtfdA. This step had the benefit of eliminating a HindII site that interfered with further cloning steps. The 727-base pairHindII-KpnI fragment of pBCtfdA was subcloned into pBC KS− cut with the same enzymes to give pHKtfdA. Similarly, the gene encoding H200A TfdA was made by mutagenesis of pXXtfdA, a pUC19 plasmid containing the 5′-XbaI-XhoI fragment of tfdA. The altered XbaI-XhoI fragment was then inserted into pHKtfdA cut with XbaI and XhoI to give the complete H200A tfdA gene. The identity of all final constructs was confirmed by sequence analysis. To insert the genes encoding H167A and H262A variants of TfdA into a plasmid that would allow for isopropyl-1-thio-β-d-galactopyranoside-controlled expression, the XbaI-SalI fragments from the corresponding plasmids described above were cloned into pET23a (Novagen) prepared with the same enzymes.Table IISequences of mutagenic primers used to create altered tfdA genesMutantPrimer sequenceaThe reverse primer used for the creation of all mutants was the complement of the forward primer.H8A5′-CGC AAA TCC CCT TGC TCC TCT TTT CGC C-3′H113A5′-GTC GCT GGC CCA AGC TGG-3′D115A5′-GCA CAG CGC CAG CTC CTT TCA-3′H167A5′-GCG TGC CGA GCA GTA CGC ACT G-3′H200A5′-GGT TCG AAC CGC CGC CGG CTC-3′H213A5′-GCT CGC GGC CGC GCC GAT-3′H216A5′-CCT TCG ACG GCG CTC GCG-3′H235A5′-GCT TCT CGA GGC GAC ACA G-3′H245A5′-GTG TAC CGG GCT CGC TGG AAC-3′H262A5′-CGT TCT TGC ACG CGG ACG CAG-3′Tfda-MBPF5′-TCT CTA GAG TGA GCG TCG TCG CAA ATC C-3′Tfda-MBPR5′-GTC AAG CTT GGT TGC GTA CAT CTT GTG G-3′a The reverse primer used for the creation of all mutants was the complement of the forward primer. Open table in a new tab To create the maltose-binding protein (MBP)-TfdA fusion proteins, the wild-type tfdA gene was amplified from pUS311 with TfdA-MBPF and TfdA-MBPR primers (Table II) to create an XbaI site directly upstream of the GTG start codon of the tfdA gene and a HindIII restriction site 54 base pairs downstream of the stop codon. The polymerase chain reaction product was cloned directly into the pGEM-T vector according to the manufacturer's instructions (Promega, Madison, WI). TheXbaI-HindIII fragment was isolated from the resulting plasmid and cloned into the pMAL-c2 vector that had been digested with the same enzymes. The identity of the newly createdmalE-tfdA gene fusion was confirmed by sequencing. Substitution of the mutation-containing internalNruI fragment for the same fragment of the wild-typemalE-tfdA gene created MBP-fusion forms of altered TfdAs. First, pMAL-tfdA was digested withNruI, and the vector fragment was purified and religated to create pMAL-tfdAΔNruI. The resultant plasmid was linearized with NruI and dephosphorylated with calf intestine alkaline phosphatase prior to ligation with theNruI fragments isolated from the previously described mutant genes. Constructs were confirmed by restriction analysis. H8A, H113A, D115A, H216A, H235A, and wild-type TfdA proteins were purified from E. coli DH5α cells carrying pUS311 and its mutated derivatives according to a previously described protocol (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar). In addition, the non-mutated enzyme and the TfdA variants H167A, H200A, H213A, H245A, and H262A were purified as MBP-TfdA fusion proteins from E. coli DH5α by the protocol described in the pMAL Protein Fusion and Purification System Manual (New England Biolabs, Beverly, MA). Specific activities of the wild-type and variant TfdA proteins were determined by a previously described spectrophotometric assay (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar). The typical assay mixture contained 1 mm 2,4-D, 1 mm α-KG, 100 μm(NH4)2Fe(SO4)2, and 100 μm ascorbic acid in 10 mm MOPS buffer (pH 6.75) at 30 °C. The reactions were quenched by the addition of EDTA to a concentration of 5 mm. 2,4-Dichlorophenol was quantified by reaction with 4-aminoantipyrene followed by measurement of the absorbance at 510 nm. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of dichlorophenol·min−1. Protein concentrations were determined using the Bio-Rad Protein Assay with bovine serum albumin as a standard. For calculation of the k cat values, the TfdA variants were assumed to have M r = 31,600 and the MBP-TfdA variants were assignedM r = 74,500. The low K m values for α-KG (∼2–5 μm for the wild-type enzyme) precluded use of the 4-aminoantipyrene assay for accurate determination of this value. The alternative method used to measure the K m values for α-KG quantified the amount of 14CO2 liberated from α-[1-14C]KG during the course of the reaction (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar). Size exclusion chromatography was used to estimate the native molecular weights of TfdA, MBP-TfdA, and mutant proteins. The proteins were chromatographed on a Superose 6 gel filtration column (1.0 × 30 cm, Amersham Pharmacia Biotech) in 20 mm Tris buffer (pH 7.5), 1 mm EDTA, and 200 mm NaCl at a flow rate of 0.2 ml·min−1. The elution volumes were compared with those for gel filtration standards (Bio-Rad) including thyroglobulin, 670 kDa; bovine gamma globulin, 158 kDa; chicken ovalbumin, 44 kDa; myoglobin, 17 kDa; and vitamin B12, 1350 Da. Proteins for electron paramagnetic resonance (EPR) and electron spin-echo envelope modulation (ESEEM) spectroscopic analyses were exchanged into 25 mm MOPS (pH 6.75) by repeated concentration and dilution in Centricon 30 (Amicon) centrifugal concentrators. The final subunit concentration was 0.5 mm for the non-fusion forms of TfdA and 0.4 mmfor the MBP-TfdA proteins. CuCl2 was added to a concentration of 450 and 350 μm, respectively. Buffered solutions of α-KG and 2,4-D were added to final concentrations of 5 mm. Glycerol was present at 40% in all samples. X-band EPR spectra were obtained at 77 K on a Bruker ESP-300E spectrometer. ESEEM data were collected on a home-built spectrometer; the microwave bridge of this instrument has been previously described in detail (25.McCracken J. Shin D.-H. Dye J.L. Appl. Mag. Res. 1992; 3: 305-316Crossref Scopus (22) Google Scholar). Data collection and analyses were controlled by a Power Computing model 200 Power PC using software written with LabView version 5.01 (National Instruments). Electron spin echoes were digitized, averaged, and integrated by a Tektronix model 620B digital oscilloscope interfaced to the spectrometer computer via an IEEE-488 bus. Two four-channel delay and gate generators (Stanford Research Systems model DG535), a Bruker BH-15 magnetic field controller, and a Hewlett-Packard model 8656B radiofrequency synthesizer were also interfaced using IEEE-488 protocol. Data were collected using a reflection cavity that employed a folded microstrip resonator (26.Lin C.P. Bowman M.K. Norris J.R. J. Magn. Reson. 1985; 65: 369-374Google Scholar). A three-pulse stimulated echo sequence (90-τ-90°-T-90°) was used. ESEEM spectra were generated by Fourier transformation of the time domain data using dead time reconstruction (27.Mims W.B. J. Magn. Reson. 1984; 59: 291-306Google Scholar). Simulations of the experimental data were performed on a Sun SparcII work station. Simulation programs were written in FORTRAN and based on the density matrix formalism developed by Mims (28.Mims W.B. Phys. Rev. B: Solid State. 1972; 5: 2409-2419Crossref Scopus (532) Google Scholar). Software for the frequency analysis of the experimental and simulated data was written in Matlab (Mathworks, Natick, MA). Related sequences were initially detected by BLAST (29.Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (71456) Google Scholar) and PSI-BLAST (20.Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) analyses. Alignments were generated with the CLUSTAL algorithm (30.Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar), and the figure was prepared using Genedoc (31.Nicholas Jr., K.B. Nicholas Jr., H.B. Deerfield D.W., II EMB News. 1997; 4: 14Google Scholar). Initially, all of the mutant genes were expressed from their pUC19-based plasmids except for those encoding H113A, H167A, and H200A TfdA, which were in pBC KS−-derived plasmids. By using the standard protocol to produce soluble, wild-type TfdA (growth at 30 °C to early stationary phase), only the H8A, H113A, D115A, H216A, and H235A variants existed as soluble proteins. All of the other TfdA variants were present as inclusion bodies even when grown at lower temperatures (22 °C), in M9 minimal medium, or in LB broth containing 660 mmsorbitol and 2.5 mm betaine (32.Blackwell J.R. Horgan R. FEBS Lett. 1991; 295: 1-12Crossref PubMed Scopus (181) Google Scholar). In addition, isopropyl-1-thio-β-d-galactopyranoside-controlled production of H167A and H262A proteins from mutant genes cloned into pET23a did not yield soluble samples even when the harvested cell pellets were suspended in buffer containing 20% glycerol to limit protein aggregation. To overcome the solubility problems for the five TfdA variants, MBP-TfdA fusion proteins were created. Wild-type TfdA and the MBP-TfdA fusion protein had essentially identical k catand very similar K m values for α-KG and 2,4-D (Table III). A slight increase in the apparent K D for Fe(II) may reflect some metal binding capacity of MBP. Since the presence of the fusion protein did not appear to greatly affect the kinetic parameters of wild-type enzyme, similar fusion proteins were created for the H167A, H200A, H213A, H245A, and H262A TfdA variants.Table IIISummary of the kinetic parameters for active TfdA variantsTfdA samplek catK mαKGK m 2,4-DK D Fe(II)min −1μmμmμmWild type442 ± 334.9 ± 0.7319.3 ± 3.72.0 ± 0.5MBP fusion411 ± 239.9 ± 1.833.7 ± 2.515.2 ± 5.4H8AaMore than 75% of the protein was present as the degradation product. The k cat was estimated from the active, full-length fraction.284 ± 6.96.5 ± 1.817.7 ± 3.81.9 ± 0.7MBP-H213A22.1 ± 2.98.3 ± 2.9318 ± 4427.7 ± 9.8H216A474 ± 476.05 ± 2.652 ± 52.4 ± 1.7H235A284 ± 259.6 ± 1.634 ± 81.4 ± 1.7a More than 75% of the protein was present as the degradation product. The k cat was estimated from the active, full-length fraction. Open table in a new tab Results from kinetic analyses of the four active mutant proteins are summarized in TableIII. H8A TfdA was soluble and active but was rapidly proteolyzed to an inactive form. By electrophoretic comparisons, the cleavage site appeared to be the same as in wild-type TfdA (between Arg-77 and Phe-78) (21.Fukumori F. Hausinger R.P. J. Biol. Chem. 1993; 268: 24311-24317Abstract Full Text PDF PubMed Google Scholar). The rate of proteolysis of H8A TfdA was enhanced compared with that seen for the wild-type enzyme despite the presence of EDTA and protease inhibitors in the purification buffer. Because purified H8A TfdA was more than 75% degraded, the catalytic rate constant was calculated with the estimated amount of intact enzyme. These calculations indicate rates and K m values similar to those for the wild-type enzyme. Similarly, the kinetic parameters for H235A TfdA were comparable to the native enzyme. In contrast, two variants exhibited differences from wild-type enzyme in their kinetic parameters. The H213A MBP-TfdA variant exhibited a 20-fold reduction ink cat and a 10-fold increase inK m for 2,4-D. In addition, H216A TfdA had a modest (2.5-fold) increase in the K m for 2,4-D and no change in catalytic rate. The other kinetic parameters for H213A MBP-TfdA and H216A TfdA (K m for α-KG andK D for ferrous ion) did not differ significantly from the wild-type values. Six soluble TfdA variants (H113A, D115A, MBP-H167A, MBP-H200A, MBP-H245A, and MBP-H262A) exhibited no activity even when assayed with elevated substrate and cofactor concentrations (10 mm α-KG, 5 mm 2,4-D, and 250 μm Fe(II)). To assess whether the inactive mutant proteins assumed conformations similar to the wild-type enzyme, their apparent molecular weights were estimated by gel filtration analysis. The observed size of wild-type TfdA was found to be 51 kDa by comparison to protein standards, suggesting that TfdA forms a compact dimer or an elongated monomer. The elution volume for both H113A and D115A corresponded exactly to wild-type TfdA indicating that these proteins are not significantly altered in their quaternary structure. MBP-TfdA eluted both in the void volume (approximately 25% of the protein) and at a position corresponding to 216 kDa (roughly 75% of the protein), suggesting that MBP-TfdA forms at least a dimer. Because each MBP-TfdA subunit is comprised of two domains separated by a 13-amino acid linker, the resultant protein may migrate with a larger apparent molecular weight. MBP-H167A and MBP-H200A samples demonstrated the same two-peak profile as MBP-TfdA but with larger proportions eluting in the void volume. MBP-H245A and MBP-H262A proteins were soluble; however, gel filtration analysis indicated the presence of only highly aggregated material eluting in the void volume. Because these mutant proteins exhibited aberrations in their folding properties, the catalytic role of His-245 and His-262, if any, could not be assessed. The metallocenter properties for selected TfdA variants were probed by EPR spectroscopy. To circumvent the problems that arise in EPR measurements of integer spin paramagnetic centers, Fe(II) was substituted with cupric ion. Although the Cu(II) form of TfdA is inactive, Cu(II) binds competitively with respect to Fe(II) (K i = 1–3 μm), and copper-substituted TfdA has been used previously to study the metal coordination environment of this enzyme in the presence and absence of substrates (22.Cosper N.J. Stålhandske C.M.V. Saari R.E. Hausinger R.P. Scott R.A. J. Biol. Inorg. Chem. 1999; 4: 122-129Crossref PubMed Scopus (25) Google Scholar, 23.Whiting A.K. Que Jr., L. Saari R.E. Hausinger R.P. Fredrick M.A. McCracken J. J. Am. Chem. Soc. 1997; 119: 3413-3414Crossref Scopus (28) Google Scholar, 24.Hegg E.L. Whiting A.K. McCracken J. Hausinger R.P. Que J., L. Biochemistry. 1999; 38: 16714-16726Crossref PubMed Scopus (70) Google Scholar). Spectral parameters of wild-type Cu(II)-TfdA, Cu(II)-TfdA + α-KG, and Cu(II)-TfdA + α-KG + 2,4-D (Fig.2 A and TableIV), agreed well with those reported previously (23.Whiting A.K. Que Jr., L. Saari R.E. Hausinger R.P. Fredrick M.A. McCracken J. J. Am. Chem. Soc. 1997; 119: 3413-3414Crossref Scopus (28) Google Scholar, 24.Hegg E.L. Whiting A.K. McCracken J. Hausinger R.P. Que J., L. Biochemistry. 1999; 38: 16714-16726Crossref PubMed Scopus (70) Google Scholar). Earlier studies of copper-substituted wild-type TfdA indicated that the metal is bound in a type 2 environment with a mixture of O and N ligands in the equatorial plane. Upon addition of α-KG and 2,4-D to the enzyme, the spectral parameters are altered to a more rhombic signal with accompanying resolution of ligand hyperfine coupling (Fig. 2 A). These results suggest that binding of the co-substrates to the enzyme leads to a better defined copper site with α-KG binding directly to the metallocenter (23.Whiting A.K. Que Jr., L. Saari R.E. Hausinger R.P. Fredrick M.A. McCracken J. J. Am. Chem. Soc. 1997; 119: 3413-3414Crossref Scopus (28) Google Scholar, 24.Hegg E.L. Whiting A.K. McCracken J. Hausinger R.P. Que J., L. Biochemistry. 1999; 38: 16714-16726Crossref PubMed Scopus (70) Google Scholar). The smallA ∥ (less than 14 mT) for the α-KG- and 2,4-D-bound sample indicates a significant distortion from planarity.Table IVSummary of the EPR spectral parameters for Cu(II)-substituted TfdA variantsg ∥A ∥g ⊥mTWild-type TfdA2.3416.32.07Wild-type TfdA/α-KG2.3615.12.07Wild-type TfdA/α-KG/2,4-D2.3812.02.09H113A2.3016.62.07H113A/α-KG2.3315.82.07H113A/α-KG/2,4-D2.3514.02.08D115A2.3017.02.06D115A/α-KG2.3016.22.062.3416.82.06D115A/α-KG/2,4-D2.3016.22.062.3416.82.06MBP-H167A2.3017.12.07MBP-H167A/α-KG/2,4-D2.3017.12.07MBP-H200A2.3017.12.072.3416.12.07MBP-H200A/α-KG/2,4-D2.3017.12.072.3416.12.07 Open table in a new tab The four inactive mutant forms of TfdA with quaternary structures similar to the corresponding wild-type protein (H113A, D115A, MBP-H167A, and MBP-H200A) were analyzed by EPR spectroscopy to assess the metal coordination environments. No significant differences between the spectra of MBP-TfdA and the non-fusion wild-type TfdA were observed (data not shown). EPR spectra of
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