Identification of a Catalytic Aspartyl Residue ofd-Ribulose 5-Phosphate 3-Epimerase by Site-directed Mutagenesis

Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of d-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-typek cat of 7.1 × 103s−1 is lowered to 3.3 × 10−4s−1 in D186A, 0.13 s−1 in D186N, and 1.1 s−1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the β-carboxyl of Asp186 facilitates catalysis by >107-fold. Relative to the wild-type enzyme, the K m ford-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate α-proton abstraction. This study is the first to identify a catalytic group of the epimerase. Guided by comparative sequence considerations, we have examined the possibility of a catalytic role of Asp186 of d-ribulose 5-phosphate epimerase by site-directed mutagenesis of the recombinant spinach enzyme. Accordingly, D186A, D186N, and D186E mutants of the epimerase were constructed, purified, and characterized; as judged by their electrophoretic mobilities the mutants are properly assembled into octamers like the wild-type enzyme. Based on the extent of internal quenching of Trp fluorescence, the conformational integrity of the wild-type enzyme is preserved in the mutants. The wild-typek cat of 7.1 × 103s−1 is lowered to 3.3 × 10−4s−1 in D186A, 0.13 s−1 in D186N, and 1.1 s−1 in D186E; as gauged by D186A, altogether lacking a functional side chain at position 186, the β-carboxyl of Asp186 facilitates catalysis by >107-fold. Relative to the wild-type enzyme, the K m ford-ribulose 5-phosphate is essentially unaltered with D186N and D186E but increased 10-fold with D186A. Apart from their impairments in epimerase activity, the mutants are unable to catalyze exchange between solvent protons and the C3 proton of substrates. This deficiency and the differential alterations of kinetic parameters among the mutants are consistent with Asp186 serving as an electrophile to facilitate α-proton abstraction. This study is the first to identify a catalytic group of the epimerase. ribulose 5-phosphate isoelectric focusing polyacrylamide gel electrophoresis xylulose 5-phosphate. Despite its participation in the ubiquitous oxidative pentose phosphate pathway and its concurrent role in the reductive pentose phosphate pathway (i.e. the Calvin cycle) of photosynthetic organisms, d-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1), which catalyzes the interconversion of Ru5P1 and Xu5P, has not been well-characterized with respect to structure-function relationships. This relative neglect is probably a consequence of low natural abundance of the epimerase and, in the case of the plant enzyme, pronounced instability. We have recently removed these barriers to detailed structural and mechanistic studies by designing a heterologous overexpression system for the gene that encodes spinach chloroplast Ru5P epimerase and by devising a facile purification scheme for the recombinant enzyme that is compatible with its lability (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar). The epimerase-catalyzed reaction probably entails a two-base mechanism with an enediolate intermediate (Fig. 1). This supposition is based on the observations that Ru5P formed from [3-2H]Xu5P was completely free of deuterium, even though deuterium was completely retained in the remaining Xu5P (2Davis L. Lee N. Glaser L. J. Biol. Chem. 1972; 247: 5862-5866Abstract Full Text PDF PubMed Google Scholar). Later findings (3Hosomi S. Nakai N. Kogita J. Terada T. Mizoguchi T. Biochem. J. 1986; 239: 739-743Crossref PubMed Scopus (5) Google Scholar) that isomerization of d-erythrose 4-phosphate to d-erythrulose 4-phosphate by Ru5P epimerase proceeded by intramolecular proton transfer, whereas the C2 proton ofd-threose-4-phosphate formed concurrently by epimerization of d-erythrose-4-phosphate was derived entirely from water, lends additional credence to a two-base mechanism. In Fig. 1, we invoke a general acid to facilitate α-proton abstraction and to stabilize the enediolate intermediate based merely on the prevalence of such groups among mechanistically characterized enzymes, which also abstract α-protons of carbon acids, inclusive of triosephosphate isomerase (4Bash P.A. Field M.J. Davenport R.C. Petsko G.A. Ringe D. Karplus M. Biochemistry. 1991; 30: 5826-5832Crossref PubMed Scopus (260) Google Scholar, 5Lodi P.J. Knowles J.R. Biochemistry. 1991; 30: 6948-6956Crossref PubMed Scopus (153) Google Scholar, 6Alagona G. Ghio C. Kollman P.A. J. Am. Chem. Soc. 1995; 117: 9855-9862Crossref Scopus (71) Google Scholar), 3-ketosteroid isomerase (7Wu Z.R. Ebrahimian S. Zawrotny M.E. Thornburg L.D. Perez-Alvarado G.C. Brothers P. Pollack R.M. Summers M.F. Science. 1997; 276: 415-418Crossref PubMed Scopus (140) Google Scholar, 8Cho H.-S. Choi G. Choi K.Y. Oh B.-H. Biochemistry. 1998; 37: 8325-8330Crossref PubMed Scopus (72) Google Scholar, 9Thornburg L.D. Hénot F. Bash D.P. Hawkinson D.C. Bartel S.D. Pollack R.M. Biochemistry. 1998; 37: 10499-10506Crossref PubMed Scopus (41) Google Scholar), and mandelate racemase (10Landro J.A. Gerlt J.A. Kozarich J.W. Koo C.W. Shah V.J. Kenyon G.L. Neidhart D.J. Fujita S. Petsko G.A. Biochemistry. 1994; 33: 635-643Crossref PubMed Scopus (112) Google Scholar, 11Mitra B. Kallarakal A.T. Kozarich J.W. Gerlt J.A. Clifton J.G. Petsko G.A. Kenyon G.L. Biochemistry. 1995; 34: 2777-2787Crossref PubMed Scopus (77) Google Scholar). The catalytically functional groups depicted in Fig. 1 have not been identified, nor have any residues of Ru5P epimerase even been assigned to the active site. We wish to rectify this situation by applying site-directed mutagenesis to the cloned spinach gene. Because three-dimensional structural information and chemical modification data of Ru5P epimerase are unavailable to guide selections of site-directed mutants, we have assessed comparative sequences and predictive secondary structural analyses for candidate catalytic residues. These assessments prompted scrutiny of Asp186. As reported herein, properties of the D186E, D186N, and D186A mutants of Ru5P epimerase are entirely compatible with a crucial catalytic role for Asp186. Materials and vendors were as follows: transketolase, triosephosphate isomerase, glycerophosphate dehydrogenase, thiamine pyrophosphate,dl-α-glycerophosphate, d-ribose-5-phosphate, Ru5P, leupeptin, phenylmethylsulfonyl fluoride,N-acetyl-l-tryptophan, D2O (99.9 atom % D), lactate dehydrogenase, phospho(enol)pyruvate, pyruvate kinase, phosphoriboisomerase, ATP, and NADH, Sigma; Pfu DNA polymerase, Stratagene; DpnI restriction endonuclease, New England Biolabs; oligonucleotide primers for polymerase chain reaction mutagenesis, Life Technologies; 4-(2-aminoethyl) benzenesulfonyl fluoride, Calbiochem; 3,3′-diaminobenzidine tetrahydrochloride dihydrate and Bio-Spin 6 columns, Bio-Rad; ultrapure urea, ICN; and molecular weight calibration kits, Amersham Pharmacia Biotech. Common laboratory reagents used in conjunction with mutagenesis, expression, and enzyme purification were procured at the highest level of purity readily available. Spinach phosphoribulokinase was prepared as described earlier (12Porter M.A. Milanez S. Stringer C.D. Hartman F.C. Arch. Biochem. Biophys. 1986; 245: 14-23Crossref PubMed Scopus (66) Google Scholar, 13Porter M.A. Stringer C.D. Hartman F.C. J. Biol. Chem. 1988; 263: 123-129Abstract Full Text PDF PubMed Google Scholar). The Ru5P epimerase mutants were constructed by use of linear polymerase chain reaction in conjunction with Pfu DNA polymerase (14Weiner M.P. Costa G.L. Schoettlin W. Cline J. Mathur E. Bauer J.C. Gene (Amst.). 1994; 151: 119-123Crossref PubMed Scopus (415) Google Scholar). Plasmid pFL506 (GenBank accession number AF070943), which encodes mature wild-type spinach Ru5P epimerase ligated as anNcoI-BamHI fragment adjacent to the tac promoter of expression vector pFL260 (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar, 15Larimer F.W. Mural R.J. Soper T.S. Protein Eng. 1990; 3: 227-231Crossref PubMed Scopus (19) Google Scholar), was used as the template for mutagenesis. The mutagenic oligonucleotide primers (for forward and reverse replication) and the encoded amino acid substitutions are shown in Table I. After replication, the polymerase chain reaction reaction mixture was treated withDpnI restriction endonuclease to cleave the methylated template DNA; unmethylated product DNA is not a substrate forDpnI. Product DNA was then ethanol-precipitated, redissolved in 10 mm Tris and 1 mm EDTA (pH 8.0), and electroporated into Escherichia coli XL 1-Blue (16Dower W.J. Miller J.F. Ragsdale C.W. Nucleic Acids Res. 1988; 16: 6127-6145Crossref PubMed Scopus (2170) Google Scholar). Plasmid template was isolated from the resulting ampR transformants and sequenced across the region of interest to confirm the desired mutation.Table IOligonucleotide primers for PCR mutagenesisaThe replacement residue is underlined.D186A5′-CCG TGG ATT GAG GTA GCT GGT GGA GTT ACT-3′3′-GGC ACC TAA CTC CAT CGA CCA CCT CAA TGA-5′181 190P W I E V A G G V TD186N5′-CCG TGG ATT GAG GTA AAT GGT GGA GTT ACT-3′3′-GGC ACC TAA CTC CAT TTA CCA CCT CAA TGA-5′P W I E V N G G V TD186E5′-CCG TGG ATT GAG GTA GAA GGT GGA GTT ACT-3′3′-GGC ACC TAA CTC CAT CTT CCA CCT CAA TGA-5′P W I E V E G G V Ta The replacement residue is underlined. Open table in a new tab An overnight culture (25 ml) of the mutant expression vector in host strain MV1190 or XL 1-Blue was grown in 2X-YT medium (17Sambrook J. Fritsch E.F. Manniatis T. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory Press, New York1989: A.3Google Scholar) containing 1% (v/v) glycerol and 50 μg/ml ampicillin at 37 °C. The culture was diluted 1:100 into the same medium and incubated for 4 h with shaking (250 rpm). Isopropyl β-d-thiogalactopyranoside was added to 0.1 mm, and the incubation was continued for an additional 3 h at 37 °C, at which time the cells were harvested by centrifugation. Mutants were purified by a protocol designed for wild-type Ru5P epimerase, which entails successive chromatography on DEAE-cellulose, hydroxyapatite, and MonoQ (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar). Processing of ∼10 g of cell paste, harvested from 1-liter cultures of transformed E. coli, typically gave 5–6 mg of the purified mutant. Final preparations (>5 mg/ml) in 10 mmdl-α-glycerophosphate, 50 mm NaCl, and 1 mm EDTA (pH 8.0) were stored at −80 °C in the absence of cryoprotectant;dl-α-glycerophosphate was present at all stages of purification to mitigate spontaneous loss of activity (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar). Progression of purifications was routinely followed by SDS-PAGE of fractions generated at each step. Because of the inherently low epimerase activity of the mutants, which was overshadowed by the far greater levels from indigenous E. coli Ru5P epimerase, assays of the initial extracts were uninformative. However, becauseE. coli and recombinant spinach Ru5P epimerase are widely separated by DEAE-cellulose in the first chromatographic step of the purification (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar), assays of epimerase activity in fractions collected from this column and at all subsequent steps could be used to monitor D186N and D186E. The paucity of activity associated with D186A precluded reliable activity measurement before concentration of pooled fractions from the final chromatographic step. Similarly to the observed behavior of the wild-type epimerase (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar), each of the mutants was resolved into two peaks by chromatography on MonoQ. The catalytic parameters of the two peaks were indistinguishable. SDS-PAGE was carried out at 15 °C and pH 8.1 on 12.5% (w/v) PhastGels, and nondenaturing PAGE was carried out at 15 °C and pH 8.8 on 8–25% (w/v) gradient PhastGels, in conjunction with a Phast System apparatus (Amersham Pharmacia Biotech). IEF under denaturing conditions was performed at 15 °C on PhastGel IEF 5-8 as described earlier (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar). Gels were stained with Coomassie Blue according to the supplier's protocol. Protein concentration was determined by a dye binding method (18Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with reagent from Bio-Rad; bovine serum albumin served as the standard for comparison. Ru5P epimerase activity was assayed spectrophotometrically at 340 nm by coupling Xu5P formation to NADH oxidation via reactions catalyzed by transketolase, triosephosphate isomerase, and glycerophosphate dehydrogenase (19Kiely M.E. Stuart A.L. Wood T. Biochim. Biophys. Acta. 1973; 293: 534-541Crossref PubMed Scopus (27) Google Scholar). Stock preparations of mutants for use in determination of catalytic parameters were centrifuged at 4 °C through a Bio-Spin column (8 × 20 mm) equilibrated with 50 mm Bicine (pH 8.0) to removedl-α-glycerophosphate. If dilutions were required before initiation of enzyme assays, the 50 mm Bicine buffer containing bovine serum albumin at 1 mg/ml was used. Final concentrations of the mutant proteins in the 120-μl assay solutions were 0.01, 0.1, and 5 mg/ml for D186E, D186N, and D186A, respectively; these amounts contrast to 1.2–1.8 ng/ml as were ample to assess wild-type enzyme. Steady-state fluorescence emission spectra were recorded with an SLM-AB2 fluorimeter; excitation and emission bandwidths were set at 4 nm. All measurements were carried out at 25 °C with excitation at 295 nm to selectively excite the indole side chains. Protein samples (0.1 mg/ml withA 295 of ∼0.01) were prepared in 50 mm Bicine (pH 8.0); 8 m urea was included for examination of denatured wild-type epimerase.N-Acetyl-l-tryptophan (5 μm,A 295 of 0.01) in 50 mm Bicine (pH 8.0) was used as a standard for fluorescence emission. Controls lacking protein were used to subtract out emission spectra were the in time for of the spectra were collected at with a spectra of samples in D2O (pH 8.0) were collected a of with data chemical were to of amino acid sequences of Ru5P from of Ru5P that were and accession for their sequences are as coli, and from and identify 25 residues the with side and Asp186 in the of residues. assessed by for of secondary B. C. 1994; PubMed Scopus Google Scholar, B. C. 1994; Google Scholar), the epimerase sequence a of and compatible with an analyses a of purity of the epimerase mutants, which with the wild-type enzyme denaturing (Fig. 3 and nondenaturing (Fig. 3 A of for the D186A and D186N in contrast to a of for wild-type and D186E as determined by denaturing IEF (Fig. 3 with the of the from to The of the wild-type enzyme, which is observed by nondenaturing IEF (1Chen Y.-R. Hartman F.C. Lu T.-Y.S. Larimer F.W. Plant Physiol. 1998; 118: 199-207Crossref PubMed Scopus (12) Google Scholar), with each of the mutants not Trp fluorescence has been examined as a of conformational integrity of the mutants (Fig. with a emission for Trp that is observed for the wild-type epimerase in the of 8 m urea, fluorescence is under nondenaturing Trp fluorescence be with the mutants. each mutant is activity to cat and K m of Ru5P m associated with of by or Asp186 in parameters of spinach recombinant Ru5P m × × × × × × × × × are under Based on assays, K m by Open table in a new tab are under Based on assays, K m by The proton of Ru5P and Xu5P, the their C3 are (Fig. A and to the epimerase-catalyzed of in D2O to be by After has been (Fig. 5 the spectra the by the C3 proton of Ru5P or Xu5P, the C3 proton of substrate is in the product by deuterium from In the spectra of 5 mm Ru5P or Xu5P were recorded before and 5 the of wild-type epimerase to the solutions at a final concentration of the spectra were unaltered by incubation of substrate for with D186N at of of proton exchange activity by the of epimerase activity, is Because of the that abstract protons to or 5-phosphate isomerase that a substrate with Ru5P epimerase and that to Ru5P, we to with of sequence with Ru5P epimerase through data were of active from to Ru5P epimerase. However, predictive secondary structural analyses that the Ru5P epimerase into an as among and Petsko G.A. Biochem. 1990; Full Text PDF PubMed Scopus Google Scholar, C. J. to Protein New Scholar, Gerlt J.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast to data for proteins with sequence to Ru5P epimerase, of sequences of the epimerase from to for mutagenesis. 8 of the 25 residues have side chains. of is in the of 3 residues that the this the of 8 of the active residues of proteins this are at the of or in between of and the N of the Petsko G.A. Biochem. 1990; Full Text PDF PubMed Scopus Google Scholar, C. D. J. B. in Molecular Press, New Google Scholar, C. J. to Protein New Scholar). Asp186 as of the two general for the abstraction of the C3 proton from Ru5P or Xu5P or the general acid to in the forward and reverse of we to the of of the β-carboxyl group and of with groups that its we characterized the D186A, D186N, and D186E mutants of Ru5P epimerase. which could be purified by the that for wild-type enzymes, the same as the wild-type enzyme denaturing and nondenaturing the mutant are which and properly into to Despite the of two residues in each of Ru5P epimerase, fluorescence emission by excitation at 295 nm is we have not determined the side chain for the quenching of Trp fluorescence, of this by the mutants that their are not relative to As judged by the of of the mutants × for D186A, × for D186N, and × for in conjunction with essentially unaltered K m for Ru5P with D186N and D186E, be that Asp186 of Ru5P epimerase a role in The 10-fold in m for Ru5P with D186A, in contrast to the wild-type with the two mutants, which binding at position 186, is consistent with an between the β-carboxyl of Asp186 and the group of as this residue served as the The levels of activity associated with D186N and D186E, with D186A, are also compatible with as an electrophile to facilitate abstraction of the α-proton from The 10-fold cat of D186E relative to that of D186N a mechanism the enediolate intermediate is by of the active proton to the of the intermediate (i.e. formation of a as in the case of mandelate (10Landro J.A. Gerlt J.A. Kozarich J.W. Koo C.W. Shah V.J. Kenyon G.L. Neidhart D.J. Fujita S. Petsko G.A. Biochemistry. 1994; 33: 635-643Crossref PubMed Scopus (112) Google Scholar, 11Mitra B. Kallarakal A.T. Kozarich J.W. Gerlt J.A. Clifton J.G. Petsko G.A. Kenyon G.L. Biochemistry. 1995; 34: 2777-2787Crossref PubMed Scopus (77) Google Scholar, J.A. J. Am. Chem. Soc. Scopus Google Scholar, J.A. Biochemistry. PubMed Scopus Google Scholar). the side chain of could the substrate and intermediate through proton is of the an If Asp186 as of the two for α-proton abstraction from D186N be as as is active the and 10 active D186E. In to of catalysis proton we examined the of D186N to catalyze exchange of solvent protons with the C3 proton of Ru5P or Xu5P by In the case of mandelate replacement of the that the α-proton from by racemase activity and exchange activity between D2O and the mutant catalyzed the exchange reaction between D2O and the α-proton of at a observed with wild-type enzyme B. Kallarakal A.T. Kozarich J.W. Gerlt J.A. Clifton J.G. Petsko G.A. Kenyon G.L. Biochemistry. 1995; 34: 2777-2787Crossref PubMed Scopus (77) Google Scholar). This of exchange activity was in the role of concentrations of the D186N mutant in D2O that have readily of exchange activity at of the were with Ru5P and Xu5P as of differential exchange activity, relative to epimerase activity, associated with D186N is in with Asp186 serving as the the in of the initial substrate step. the to of a to the exchange of deuterium from D2O into substrate the to an enzyme exchange with solvent the substrate enzyme This not be the of [3-2H]Xu5P in by wild-type Ru5P epimerase, deuterium is not from the remaining of substrate (2Davis L. Lee N. Glaser L. J. Biol. Chem. 1972; 247: 5862-5866Abstract Full Text PDF PubMed Google Scholar). the proton exchange with solvent the enediolate intermediate at the active or the of the intermediate by the to product is far exchange of protons between the first and In we have that Asp186 of Ru5P epimerase and to Asp186 as the electrophile that facilitates α-proton abstraction by of the general but its as of the two be studies of solvent and substrate this We the of S. Lu and