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

Yuh-Ru ChenDagger , Frank W. Larimer§, Engin H. Serpersuparallel , and Fred C. Hartman§

From the § Protein Engineering Program, Life Sciences Division, Oak Ridge National Laboratory, and Dagger  University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37831 and parallel  Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996

    ABSTRACT
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Abstract
Introduction
References

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-type kcat of 7.1 × 103 s-1 is lowered to 3.3 × 10-4 s-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 beta -carboxyl of Asp186 facilitates catalysis by >107-fold. Relative to the wild-type enzyme, the Km for D-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 alpha -proton abstraction. This study is the first to identify a catalytic group of the epimerase.

    INTRODUCTION
Top
Abstract
Introduction
References

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 (1).

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 (2). Later findings (3) that isomerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate by Ru5P epimerase proceeded by intramolecular proton transfer, whereas the C2 proton of D-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 alpha -proton abstraction and to stabilize the enediolate intermediate based merely on the prevalence of such groups among mechanistically characterized enzymes, which also abstract alpha -protons of carbon acids, inclusive of triosephosphate isomerase (4-6), 3-ketosteroid isomerase (7-9), and mandelate racemase (10, 11).


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Fig. 1.   Likely reaction pathway for the Ru5P epimerase catalyzed interconversion of Ru5P and Xu5P. R1 = -CH2OPO32-; R2 = -HC(OH)-CH2OPO32-.

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.

    EXPERIMENTAL PROCEDURES

Materials-- Materials and vendors were as follows: transketolase, triosephosphate isomerase, glycerophosphate dehydrogenase, thiamine pyrophosphate, DL-alpha -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 (12, 13).

Site-directed Mutagenesis and Expression of Ru5P Epimerase Mutants-- The Ru5P epimerase mutants were constructed by use of linear polymerase chain reaction in conjunction with Pfu DNA polymerase (14). Plasmid pFL506 (GenBank accession number AF070943), which encodes mature wild-type spinach Ru5P epimerase ligated as an NcoI-BamHI fragment adjacent to the tac promoter of expression vector pFL260 (1, 15), 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 with DpnI restriction endonuclease to cleave the methylated template DNA; unmethylated product DNA is not a substrate for DpnI. 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 (16). Plasmid template was isolated from the resulting ampR transformants and sequenced across the region of interest to confirm the desired mutation.

                              
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Table I
Oligonucleotide primers for PCR mutagenesisa

An overnight culture (25 ml) of the mutant expression vector in host strain MV1190 or XL 1-Blue was grown in 2X-YT medium (17) 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 beta -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.

Purification of Recombinant Ru5P Epimerase Mutants-- Mutants were purified by a protocol designed for wild-type Ru5P epimerase, which entails successive chromatography on DEAE-cellulose, hydroxyapatite, and MonoQ (1). 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 mM DL-alpha -glycerophosphate, 50 mM NaCl, and 1 mM EDTA (pH 8.0) were stored at -80 °C in the absence of cryoprotectant; DL-alpha -glycerophosphate was present at all stages of purification to mitigate spontaneous loss of activity (1).

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, because E. coli and recombinant spinach Ru5P epimerase are widely separated by DEAE-cellulose in the first chromatographic step of the purification (1), 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 (1), each of the mutants was resolved into two peaks by chromatography on MonoQ. The catalytic parameters of the two peaks were indistinguishable.

Electrophoresis-- 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 (1). Gels were stained with Coomassie Blue according to the supplier's protocol.

Protein and Enzyme Assays-- Protein concentration was determined by a dye binding method (18) 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 (19). 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 remove DL-alpha -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.

Fluorescence Measurements-- 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 with A295 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, A295 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 background signals. Recorded emission spectra were automatically corrected, because the instrument accounts in real time for wavelength-dependent efficiencies of the light source.

NMR Spectroscopy-- NMR spectra were collected at 300K with a wide-bore Bruker AMX 400 MHz spectrometer. One-dimensional 1H NMR spectra of samples in D2O (pH 8.0) were collected over a spectral width of 4807 Hz with 32K data points. Proton chemical shifts were referenced to external disodium 2,2-dimethyl-2-silapentane-5-sulfonate.

    RESULTS

Comparative Sequences and Predictive Secondary Structure of Ru5P Epimerase-- Comparisons of DNA-deduced amino acid sequences of Ru5P epimerases from 16 species,2 including representatives from prokaryotes, eukaryotes, and archaea, identify only 25 invariant residues (Fig. 2). Among the eight with ionizable side chains (Asp39, Asp44, Asp47, Asp186, His42, His75, His99, and Glu101), Asp186 occurs in the only segment of contiguous invariant residues. When assessed by neural networks for predictions of secondary structure (20, 21), the epimerase sequence describes a series of alternating beta -strands and alpha -helices, compatible with an eight-stranded beta /alpha barrel folding motif.


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Fig. 2.   Primary structure of Ru5P epimerase. Species invariant residues are denoted by boldface. Assignment of residues to beta -strands (E) or alpha -helices (H) is based on predictive secondary structure analysis (20, 21). The N-terminal Ala is a consequence of the expression construct used to produce the recombinant enzyme (1); the authentic enzyme isolated from spinach chloroplast begins with Thr (28), which is therefore designated residue 1.

Purity and Structural Integrity of Ru5P Epimerase Mutants-- Electrophoretic analyses indicate a high degree of purity of the epimerase mutants, which comigrate with the wild-type enzyme during denaturing (Fig. 3A) and nondenaturing (Fig. 3B) PAGE. A pI of 6.6 for the D186A and D186N subunits in contrast to a pI of 6.3 for wild-type and D186E subunits, as determined by denaturing IEF (Fig. 3C), agrees with the calculated impact of changing the net charge from -4 to -3. The uncharacterized microheterogeneity of the wild-type enzyme, which is observed by nondenaturing IEF (1), persists with each of the mutants (data not shown).


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Fig. 3.   Electrophoretic comparisons of wild-type and mutant Ru5P epimerases by SDS-PAGE (A), nondenaturing PAGE (B), and denaturing IEF (C). The molecular mass markers (from top to bottom) in A are phosphorylase b, 94 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 20.1 kDa; and lactalbumin, 14.4 kDa. The molecular mass markers (from top to bottom) in B are thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase 232 kDa; lactate dehydrogenase, 140 kDa; and bovine serum albumin, 67 kDa. The pH gradient (8-5) in C decreases from top to bottom.

Trp fluorescence has been examined as a diagnostic of conformational integrity of the mutants (Fig. 4). Compared with a typical emission spectrum for Trp that is observed for the wild-type epimerase in the presence of 8 M urea, fluorescence is almost totally quenched under nondenaturing conditions. Likewise, little Trp fluorescence can be detected with the nondenatured mutants.


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Fig. 4.   Corrected fluorescence emission spectra of wild-type and mutant Ru5P epimerases. Spectra were collected at 2 nm/s; additional details are provided under "Experimental Procedures." w.t., wild type.

Catalytic Properties of Ru5P Epimerase Mutants-- Although each mutant is severely impaired catalytically, sufficient activity remains to quantify kcat and Km of Ru5P (Table II). Only slight increases in Km associated with substitution of Asp by Asn or Glu implicate Asp186 directly in catalysis.

                              
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Table II
Kinetic parameters of spinach recombinant Ru5P epimerase
Experimental details are provided under "Experimental Procedures." Based on replicate assays, Km and Vmax values vary by ±10%.

The proton resonances of Ru5P and Xu5P, including the doublets reflecting their respective C3 protons, are sufficiently different (Fig. 5, A and B) to allow the epimerase-catalyzed conversion of either compound in D2O to be monitored by 1H NMR. After equilibrium has been reached (Fig. 5C), the spectra lack the doublet given by the C3 proton of Ru5P or Xu5P, because the C3 proton of substrate is replaced in the product by deuterium from solvent. In the experiments depicted, spectra of 5 mM Ru5P or Xu5P were recorded before and 5 min after the addition of wild-type epimerase to the respective solutions at a final concentration of 2.2 µg/ml. By contrast, the spectra were unaltered by incubation of either substrate for 30 min with D186N at 28 µg/ml. Thus, evidence of preferential retention of proton exchange activity by the mutant, independent of epimerase activity, is lacking.


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Fig. 5.   Proton NMR spectra of Ru5P (A), Xu5P (B), and Ru5P or Xu5P after incubation with wild-type Ru5P epimerase (C). Only one spectrum is shown for the two incubations, because the spectra generated were superimposable. Assignment of protons is designated by the numbered carbon atoms (C1-C5) to which they are bonded. Additional details are provided under "Experimental Procedures."


    DISCUSSION

Because of the numerous enzymes that abstract protons alpha  to carbonyl or carboxyl groups, including D-ribose 5-phosphate isomerase that shares a common substrate with Ru5P epimerase and several others that transform substrates structurally similar to Ru5P, we expected to uncover some enzymes with regions of sequence similarity with Ru5P epimerase through data base searches. Somewhat surprisingly, none were found, thereby precluding prospects of extrapolating known active site features from functionally similar enzymes to Ru5P epimerase. However, predictive secondary structural analyses did suggest that the Ru5P epimerase subunit folds into an eight-stranded beta /alpha barrel, as commonly found among isomerases, racemases, and epimerases (22, 24, 25).

In contrast to data base searches for proteins with sequence similarities to Ru5P epimerase, consideration of sequences of the epimerase from evolutionarily diverse sources proved instructive to selecting targets for mutagenesis. Only 8 of the 25 invariant residues have ionizable side chains. One of these, Asp186, is located in the only stretch of 3 contiguous invariant residues that occurs throughout the entire 235-residue polypeptide chain. Furthermore, this location represents the C-terminal end of beta -strand 8 of the predicted beta /alpha barrel; most active site residues of proteins having this motif are positioned at the C-terminal ends of beta -strands or in interconnecting loops between C termini of beta -strands and the N termini of the following helices (22-24).

Envisioning Asp186 as one of the two prospective general bases for the abstraction of the C3 proton from either Ru5P or Xu5P or the prospective general acid to assist deprotonation in both the forward and reverse directions of catalysis, we wished to ascertain the consequences of deletion of the aspartyl beta -carboxyl group and of substitution with groups that might partially fulfill its normal function. Thus, we characterized the D186A, D186N, and D186E mutants of Ru5P epimerase.

Each mutant, which could be purified by the regimen that proved successful for wild-type enzymes, displays the same mobility as the wild-type enzyme during both denaturing and nondenaturing PAGE; thus, the mutant subunits are full-length translation products, which fold and assemble properly into octameric structures akin to wild type. Despite the presence of two tryptophanyl residues (Trp40 and Trp182) in each subunit of Ru5P epimerase, fluorescence emission elicited by excitation at 295 nm is barely discernible. Although we have not determined the side chain interactions responsible for the virtually complete quenching of Trp fluorescence, retention of this striking feature by the mutants indicates that their conformations are not perturbed substantially relative to wild type.

As judged by the severe impairment of kcat values of the mutants (2 × 107-fold for D186A, 5.5 × 104-fold for D186N, and 6.5 × 103-fold for D186E), in conjunction with essentially unaltered Km values for Ru5P with D186N and D186E, there can be little doubt that Asp186 of Ru5P epimerase plays a critical role in catalysis. The 10-fold increase in the Km for Ru5P with D186A, in contrast to the wild-type values with the other two mutants, which retain hydrogen binding potential at position 186, is consistent with an interaction between the beta -carboxyl of Asp186 and the carbonyl group of bound substrate, as would occur if this residue served as the electrophilic catalyst. The much higher levels of activity associated with D186N and D186E, compared with D186A, are also compatible with Asp186 functioning as an electrophile to facilitate abstraction of the alpha -proton from substrate. The 10-fold greater kcat of D186E relative to that of D186N might reflect a mechanism whereby the enediolate intermediate is normally stabilized by partial transfer of the active site carboxyl proton to the carbonyl of the intermediate (i.e. formation of a short, strong hydrogen bond), as demonstrated in the case of mandelate racemases (10, 11, 26, 27). Whereas the side chain of Asn could engage the substrate and intermediate through hydrogen bonding, partial proton transfer is unlikely because of the much higher pKa of an amide. If Asp186 rather serves as one of the two bases responsible for alpha -proton abstraction from substrates, D186N should be as debilitated as D186A; however, it is 400 times more active than the latter and only 10 times less active than D186E.

In attempts to further distinguish possible roles of Asp186 in electrophilic catalysis versus proton abstraction, we examined the ability of D186N to catalyze exchange of solvent protons with the C3 proton of Ru5P or Xu5P by 1H NMR. In the case of mandelate racemase, replacement of His297, the base that abstracts the alpha -proton from (R)-mandelate, by Asn eliminates both racemase activity and exchange activity between D2O and (R)-mandelate; however, the H297N mutant catalyzed the exchange reaction between D2O and the alpha -proton of (S)-mandelate at a rate only 3.3-fold less than observed with wild-type enzyme (11). This observation of stereospecific exchange activity was indeed instrumental in assigning the role of His297. At concentrations of the D186N mutant in D2O that would have readily allowed detection of exchange activity at 0.1% of wild type, the results were totally negative with both Ru5P and Xu5P as substrate. Lack of differential exchange activity, relative to overall epimerase activity, associated with D186N is in accord with Asp186 serving as the electrophile, rather than the base, in facilitation of the initial substrate deprotonation step.

Beyond the inherent limitations to interpretation of negative results, a particular caveat pertains to the NMR-based exchange assays. Incorporation of deuterium from D2O into substrate can only occur if the substrate-derived proton, transferred to an enzyme monoprotic base, can exchange with solvent while the deprotonated substrate remains enzyme bound. This precondition may not be met. During the turnover of [3-2H]Xu5P in H2O by wild-type Ru5P epimerase, deuterium is not lost from the remaining pool of unprocessed substrate (2). Thus, either the substrate-derived proton cannot exchange with solvent while the enediolate intermediate resides at the active site or the protonation of the intermediate by the second base to form product is far more rapid than exchange of protons between the first base and solvent.

In conclusion, we have offered rather compelling evidence that Asp186 of Ru5P epimerase contributes directly and profoundly to catalysis. Circumstantial evidence favors Asp186 as the electrophile that facilitates alpha -proton abstraction by either of the general base catalysts, but its identity as one of the two bases cannot be rigorously excluded. Future studies of solvent and substrate isotope effects may clarify this issue.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Tse-Yuan S. Lu and Alice A. Hardigree.

    FOOTNOTES

* This work was supported by the Office of Biological and Environmental Research, United States Department of Energy, under Contract DE-AC05-96OR22464 with Lockheed Martin Energy Research Corp.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Contributed equally to this research. To whom correspondence should be addressed: Life Sciences Division, Oak Ridge National Laboratory, P. O. Box 2009, Oak Ridge, TN 37831-8080. Tel.: 423-574-0959; Fax: 423-574-0793; E-mail: ffh{at}ornl.gov (F. C. H.) or fwl{at}ornl.gov (F. W. L.).

The abbreviations used are: Ru5P, ribulose 5-phosphate; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; Xu5P, xylulose 5-phosphate.

2 Species of Ru5P epimerases that were compared and GenBank accession numbers for their sequences are as follows: Caenorhabditis elegans, U28991; Saccharomyces cerevisiae, P46969; Schizosaccharomyces pombe, Z98979; E. coli, P32661; Serratia marcescens, P45455; Hemophilus influenzae, P44756; Alcaligenes eutrophus, P40117; Spinacia oleracea, AF070943; Solanum tuberosum, Q43157; Synechocystis sp, P74061; Bacillus subtilis, Y13937; Rhodobacter capsulatus, U23145; Mycobacterium tuberculosis, P71676; Helicobacter pylori, P56188; Rhodospirillum rubrum, P51013; and Methanococcus jannashii, Q58093.

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