Pyruvate Formate-Lyase-Activating Enzyme - Chemistry and ...

Biochemical and Biophysical Research Communications 269, 451– 456 (2000) doi:10.1006/bbrc.2000.2313, available online at http://www.idealibrary.com on

Pyruvate Formate-Lyase-Activating Enzyme: Strictly Anaerobic Isolation Yields Active Enzyme Containing a [3Fe– 4S] ⫹ Cluster Joan B. Broderick, 1 Timothy F. Henshaw, Jennifer Cheek, Kristi Wojtuszewski, Sheila R. Smith, Matthew R. Trojan, 2 Ryan M. McGhan, 2 Amy Kopf, Megan Kibbey, and William E. Broderick Department of Chemistry, Michigan State University, East Lansing, Michigan 48824

Received January 28, 2000

Pyruvate formate-lyase-activating enzyme (PFL-AE) from Escherichia coli (E. coli) catalyzes the stereospecific abstraction of a hydrogen atom from Gly734 of pyruvate formate-lyase (PFL) in a reaction that is strictly dependent on the cosubstrate S-adenosyl-Lmethionine (AdoMet). Although PFL-AE is an irondependent enzyme, isolation of the enzyme with its metal center intact has proven difficult due to the oxygen sensitivity and lability of the metal center. We report here the first isolation of PFL-AE under nondenaturing, strictly anaerobic conditions. Iron and sulfide analysis as well as UV–visible, EPR, and resonance Raman data support the presence of a [3Fe– 4S] ⴙ cluster in the purified enzyme. The isolated native enzyme, but not apo-enzyme, exhibits a high specific activity (31 U/mg) in the absence of added iron, indicating that the native cluster is necessary and sufficient for enzymatic activity. © 2000 Academic Press

Pyruvate formate-lyase (PFL) catalyzes the first committed step in anaerobic glucose metabolism in E. coli, the conversion of pyruvate and CoA to formate and acetyl-CoA, in a reaction that is strictly dependent on the presence of a radical on a glycine residue of PFL (1–3). This unusual protein main-chain centered radical has been identified and localized to Gly734 by a combination of isotopic labeling and analysis of the products of oxygenolytic cleavage of the radicalcontaining PFL (4). The radical is generated by the stereospecific abstraction of the pro-S hydrogen atom of PFL Gly734 (5). Further work by Kozarich and coworkers has provided evidence that this glycyl radical serves to generate, via hydrogen atom abstraction, a 1

To whom correspondence should be addressed at Department of Chemistry, 414A Chemistry Building, Michigan State University, East Lansing, MI 48824. Fax: (517) 353-1793. E-mail: broderij@ cem.msu.edu. 2 Undergraduate research participants, Amherst College.

thiyl radical in the active site of PFL that is directly involved in catalysis (6, 7). The catalytically essential glycyl radical of PFL is posttranslationally generated under anaerobic conditions by a specific activating enzyme, the pyruvate formate-lyase-activating enzyme (PFL-AE, Scheme 1) (8 –10). PFL-AE utilizes S-adenosylmethionine (AdoMet) as an obligate cosubstrate in the radical generation reaction, converting it stoichiometrically to methionine and 5⬘-deoxyadenosine (5⬘-dAdo) (2). In elegant experiments by Knappe and co-workers, the hydrogen atom abstracted from Gly734 of PFL has been shown to be stoichiometrically incorporated into the 5⬘-dAdo product, thereby implicating an intermediate adenosyl radical as the immediate hydrogen atom abstractor (5). PFL-AE was first isolated by Knappe and co-workers from nonoverexpressing E. coli cells (1). The enzyme was isolated aerobically and shown to be a 28 kDa monomer, and it exhibited an absorption spectrum that was attributed to an unidentified covalently bound cofactor. The enzymatic activity of PFL-AE was reported to be completely dependent on the presence of exogenous iron in the assay. Kozarich and co-workers reported the first purification and characterization of PFL-AE overexpressed in E. coli, but solubility problems required resorting to purification of guanidinedenatured protein, followed by refolding (11). The resulting apo-enzyme was found to bind stoichiometric quantities of iron or other divalent metals. Enzymatic activity was observed in the presence of Fe(II) and DTT, but thiophilic metals such as Cu(II), Zn(II), Hg(II), and Cd(II) were found to be inhibitors of PFLAE. The proposed cysteinal coordination environment was supported by early spectroscopic data from our laboratory, which provided evidence for an iron–sulfur cluster in PFL-AE (12). We report here for the full details of the first isolation of PFL-AE in its native state under strictly anaerobic conditions, and the characterization of the iron-

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SCHEME 1

sulfur cluster present in the isolated native enzyme. The ability to isolate large quantities of native PFLAE, without resorting to denaturation and/or artificial reconstitution, is critical to understanding the nature of the cluster and its role in radical generation. MATERIALS AND METHODS The plasmids pMG-AE and pKK-PFL were obtained as a generous gift from John Kozarich (Merck). The Escherichia coli BL21(DE3)pLysS strain and pCAL-n-EK expression vector were obtained from Stratagene. 5⬘-Deazariboflavin was synthesized in our laboratory according to published procedures (13–15), and characterized using NMR and TLC. All other chemicals were obtained commercially and used as received. Growth and expression of PFL. pKK-PFL was used to transform BL21(DE3)pLysS. A single colony of transformed cells was used to inoculate 5 to 50 mL of LB media containing 50 ␮g/mL ampicillin (LB/Amp). This culture was grown to saturation and then used to inoculate LB/Amp in 2.8 L Fernbach flasks or in a 10-L bench-top fermentor (New Brunswick). The cultures were grown at 37°C with vigorous shaking (flasks) or continuous air purge and vigorous agitation (fermentor) to early log phase (OD 600 ⬃ 0.6 – 0.8), and then induced by addition of isopropyl-␤-D-thiogalactopyranoside (IPTG) to 1 mM. The cultures were grown for 2 more h before harvesting by centrifugation (8000 rpm, Sorvall GS3 rotor). The supernatant was decanted and the cells stored at ⫺80°C. Purification of pyruvate formate-lyase. Pyruvate formate-lyase (PFL) was purified from the BL21(DE3)pLysS/pKK-PFL cells. Cells from 1 L of liquid culture, grown and expressed as described above, were suspended in 20 mL of enzymatic lysis buffer containing 20 mM Hepes, pH 7.2, 5 mM DTT, 1% Triton X-100, 5% (w/v) glycerol, 10 mM MgCl 2, 8 mg lysozyme, 1 mM PMSF, and trace quantities (approximately 0.1 mg each) RNase A and DNase I. The suspension was agitated with a pipet and then incubated on ice for 1 h. The lysed cells were centrifuged at 15,000 rpm (SS34) for 20 min at 4°C. The extract was decanted and either used directly in purification or flash-frozen and stored at ⫺80°C. A 10 mL portion of the crude extract was loaded onto a Q-Sepharose column (2.6 ⫻ 20 cm) equilibrated with Buffer A (20 mM Hepes, pH 7.2, 5 mM DTT). The column was washed with 50 mL of the same buffer prior to running a gradient from Buffer A to Buffer B (20 mM Hepes, 500 mM NaCl, 5 mM DTT, pH 7.2) over 100 mL. Under these conditions PFL eluted at approximately 0.25 M NaCl. Fractions containing ⱖ75% pure PFL (as judged by SDS-PAGE) were combined, flash frozen, and stored at ⫺80°C. Another 10 mL aliquot of crude extract was run through the same procedure, and the ⱖ75% pure fractions from both runs were combined, dialyzed against Buffer A, and re-run on the Q-Sepharose column as described above. The fractions containing ⱖ95% pure PFL were combined, concentrated, flash-frozen, and stored at ⫺80°C. Subcloning, growth, and expression of PFL-AE. The pflA gene, which encodes PFL-AE, was cut out of pMG-AE using NdeI/HindIII

and ligated to pCAL-n-EK that had been digested with the same enzymes. This vector was used to transform BL21(DE3)pLysS. A single colony of the resulting overexpressing strain was used to inoculate 50 mL of LB containing 50 ␮g/mL ampicillin (LB/Amp). This culture was grown to saturation at 37°C and then used to inoculate 10 L of LB/Amp. The 10 L culture was grown at 37°C in a bench-top fermentor (New Brunswick) with a continuous air purge and vigorous agitation. When the culture reached an optical density of 0.6 to 0.8, isopropyl-␤-D-thiogalactopyranoside (IPTG) was added to 1 mM final concentration, and the medium was supplemented with 150 mg Fe(NH 4) 2(SO 4) 2 per liter of culture. The culture was allowed to grow for an additional 2 h, at which time the temperature was reduced to 4°C and the culture was purged with nitrogen. The culture was incubated for 14 –24 h at 4°C under nitrogen before harvesting under anaerobic conditions. The harvested cells were stored under nitrogen at ⫺80°C until used for purification. Purification of pyruvate formate-lyase-activating enzyme (PFL-AE). PFL-AE was purified from E. coli BL21(DE3)pLysS transformed with pCAL-n-AE3, prepared as described above. All steps in the purification were performed in a single day under strictly anaerobic conditions in a Coy anaerobic chamber (Coy Laboratories, Grass Lake, MI) at ambient temperature except where noted. Solutions and buffers used in the purification were thoroughly degassed prior to bringing them in to the Coy chamber. Approximately 4 to 6 g of cell paste was suspended in 5 to 10 mL of enzymatic lysis buffer containing 50 mM Tris–sulfate, pH 7.5, 200 mM NaCl, 1% Triton X-100, 5% glycerol, 10 mM MgCl2, 1 mM DTT, 40 ␮M dithionite, 1 mM PMSF, 8 mg lysozyme, and trace amounts (approximately 0.1 mg each) of DNase I and RNase A. This suspension was incubated at ambient temperature for 1 h, and then centrifuged at 15,000 rpm (SS34) for 15 min at 4°C. The extract was decanted and used directly in purification. Up to 10 mL of the crude extract was loaded onto a Sephacryl S-200 HR column (5 ⫻ 60 cm) equilibrated with 50 mM Hepes, 200 mM NaCl, pH 7.2. The protein was eluted with this same buffer at 3 mL/min. AE eluted from the column in a relatively sharp peak at approximately 750 mL after injection. The fractions were analyzed by SDS-PAGE, and those determined to be ⱖ95% pure were pooled and concentrated using an Amicon concentrator with YM10 filter membranes. If additional purification was required, the protein was chromatographed on a Superdex 75 column (1.6 ⫻ 60 cm) using the same buffers. The concentrated, purified protein was flash-frozen and stored in O-ring-sealed tubes at ⫺80°C. Protein, iron, and sulfide assays. Routine determinations of protein concentrations were done by the method of Bradford (16), using a kit sold by Bio-Rad, and bovine serum albumin as a standard. Calibration of the results from the Bradford assays was obtained by amino acid hydrolysis of the purified enzymes, performed at the MCB Core Facility, University of Massachusetts, Amherst. Actual protein concentrations could then be determined by applying a correction factor to the Bradford assays. Iron assays were carried out by using the method of Beinert (17). Sulfide assays were carried out with a modification of the method of Beinert (18). The use of siliconized Eppendorf tubes was found to yield more reproducible results, perhaps due to the minimized head space for loss of sulfide as H2S. The tubes were kept tightly capped except when adding reagents. Rather than using stir bars, the tubes were closed and vortexed when mixing was called for. The procedure used was as follows. The sample volumes were brought to 100 ␮L with pH 8 water. One at a time, each tube was opened, 300 ␮L 1% ZnOAc and 15 ␮L 12% NaOH were added simultaneously, the tube was closed tightly and vortexed. When all tubes had been treated in this way, they were allowed to sit for 12 to 15 h before addition (again, one tube processed at a time) of 75 ␮L DMPD (0.1% in 5 N HCl) and 2 ␮L FeCl 3 (23 mM in 1.2 N HCl). Using Na2S 䡠 9H 2O as a standard as described by Beinert, this method proved very reproducible in our hands. Assay of PFL-AE activity. The activity of PFL-AE was assayed using modifications of the coupled enzyme assays previously published (1, 19). The PFL-AE reaction mix contained in a final volume

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of 450 ␮L: 0.15 M Tris chloride, pH 7.6, 0.1 M KCl, 10 mM oxamate, 8 mM DTT, 0.1 to 0.4 ␮g PFL-AE, 80 to 150 ␮g PFL, 0.2 mM AdoMet, and 50 ␮M 5⬘-deazariboflavin. This mix was made in the anaerobic chamber by combining reagents from anoxic stock solutions in the order listed to the final concentrations indicated. In all cases, 5⬘deazariboflavin addition occurred last, in the dark, and the reaction was initiated by illumination of the sample with a 300 W halogen bulb. The samples were maintained at constant temperature (24 – 26°C) during illumination by immersion in a water bath. After a specified period of illumination, typically 5 to 20 min, the samples were wrapped in aluminum foil and an aliquot was removed for assay of active PFL through the coupling assay. The coupling assay mix contained 0.1 M Tris chloride, pH 8.1, 3 mM NAD ⫹, 55 ␮M CoA, 0.1 mg BSA, 10 mM pyruvate, 10 mM malate, 20 units citrate synthase, 300 units malic dehydrogenase, and 10 mM DTT. All reagents except the DTT were combined and made anaerobic by freeze–pump–thaw cycles. The DTT was added after bringing the anaerobic mix into the anaerobic chamber. To assay PFL activity, 990 ␮L of this mix and 10 ␮L of the activated PFL described above were combined in a quartz cuvette, which was then sealed with a septum and brought out of the chamber to monitor the production of NADH by the increase in absorbance at 340 nm. One international unit of PFL-AE activity is defined as the amount that catalyzes the production of 1 nmol of active PFL per min (20). One unit of PFL activity catalyzes the production of one ␮mole of pyruvate per min, and 35 units of PFL activity correspond to 1 nmol of PFL active sites. Spectroscopic measurements. UV-Visible spectra were recorded on an HP8453 diode-array spectrophotometer equipped with a thermostated cell maintained at 4°C. EPR measurements were obtained at X-band on a Bruker ESP300E spectrometer equipped with a liquid He cryostat and a temperature controller from Oxford Instruments. Resonance Raman spectroscopy was performed by using a stilbene dye laser (Coherent) pumped with a Spectra Physics Argon Ion Laser, and a SPEX triple-grating monochromator equipped with a PAR CCD detector. The sample was contained in a spinning cell cooled to ⬍10°C by a stream of nitrogen.

RESULTS Overexpression and purification of PFL and PFL-AE. PFL was purified from BL21(DE3)pLysS cells harboring the pKK-PFL plasmid. The cells were lysed by an enzymatic procedure in a modified low-salt buffer containing 5 mM DTT. Two portions of partially purified PFL from the first ion-exchange column were combined, dialyzed, and re-run on the ion-exchange column to yield ⱖ95% pure PFL. Typical yields were 100 –120 mg of purified PFL from one liter of bacterial culture. The pflA gene was subcloned into pCAL-n-EK in order to provide an IPTG-inducible expression system and thus eliminate the need for heat-inducible expression. The resulting PFL-AE expression vector, pCALn-AE3, was used to transform BL21(DE3)pLysS for overproduction of PFL-AE. The overexpressing cells were lysed by using an enzymatic lysis procedure, and PFL-AE was successfully purified from this crude extract by one passage through a preparative gel filtration column. The enzyme eluted as a reddish-brown peak, and pure fractions were identified by SDS– PAGE, combined, concentrated, and stored under nitrogen in small aliquots at ⫺80°C. Occasionally, a

FIG. 1. UV–visible absorption spectrum of pyruvate formatelyase-activating enzyme as isolated. The protein was 0.34 mM in 50 mM Hepes/200 mM NaCl, pH 7.2, and the spectra were recorded in a 1 mm pathlength cuvette under anaerobic conditions at 4°C.

small amount of contaminating species was found to contribute to the visible absorbance of the protein at 425 nm. When this was found, PFL-AE was further purified by passage over a Superdex 75 column. Typical yields were 70 –90 mg protein per 8 g of cell paste. Enzymatic activity. For our current preparations of PFL and PFL-AE, we can achieve specific activities of 31 ⫾ 3 U/mg (at 25°C) in the absence of any added iron in either the PFL-AE activation mix or in the PFL coupling assay mix. Specific activities of 43 ⫾ 5 U/mg (at 25°C) have been achieved with 0.2 mM Fe(II) in the activation mix. Characterization of purified PFL-AE. Purified PFL-AE contains iron (2.8 ⫾ 0.3 mol/mol PFL-AE) and acid-labile sulfide (2.9 ⫾ 0.2 mol/mol PFL-AE). The UV-visible spectrum of purified PFL-AE is indicative of the presence of an iron sulfur cluster (Fig. 1), with a maximum at 412 nm (⑀ ⫽ 3.0 mM ⫺1cm ⫺1), and shoulders at 320 nm (⑀ ⫽ 4.4 mM ⫺1cm ⫺1), 455 nm (⑀ ⫽ 2.6 mM ⫺1cm ⫺1), and 550 nm (⑀ ⫽ 0.94 mM ⫺1cm ⫺1). The energies and extinction coefficients of these absorption bands are similar to those observed for the cuboidal [3Fe– 4S] ⫹ form of aconitase (21). PFL-AE exhibits a strong, nearly isotropic electron paramagnetic resonance (EPR) signal which is centered at g ⫽ 2.02 and observable only below approximately 30 K (Fig. 2). The g value and low anisotropy of this fast-relaxing signal are consistent with its assignment to a [3Fe– 4S] ⫹ cluster (22). The signal, quantified by using a Cu(II)edta standard and the method outlined by Aasa and Va¨nngård (23), accounts for 62% of the total iron in the sample, with the remainder of the iron being present in an EPR-silent form. Resonance Raman spectroscopy of the as-isolated enzyme (Fig. 3) corroborates the EPR results. The asisolated PFL-AE exhibits well-resolved peaks at 343, 378, and 417 cm ⫺1, as well as broad peaks at 270 and

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FIG. 2. X-band EPR spectra of pyruvate formate-lyase-activating enzyme as a function of temperature. PFL-AE is 1.26 mM in 50 mM Hepes/200 mM NaCl, pH 7.2. Conditions of measurement, T as indicated, microwave power, 0.02 mW, microwave frequency, 9.4792 GHz, modulation amplitude, 10.084, and receiver gain, 2 ⫻ 10 4, 5 scans accumulated for each spectrum.

the cluster site in the isolated enzyme. When apoenzyme is assayed in the absence of exogenous iron, no activity is observed. Our results provide the first unequivocal evidence that the cluster present in native isolated PFL-AE is the catalytically essential metal center for generation of the glycyl radical of PFL. The originally reported assay procedures for PFL-AE and PFL included 0.2 mM Fe(II) in both assays, and this was believed to be an absolute requirement for PFL-AE activity. Ku¨lzer et al. have recently shown that apo PFL-AE can be reconstituted with iron and sulfide, and that the enzyme is then active in the absence of added iron in the assay (20). The details of their report, however, indicate that full activity in the absence of added iron is achieved only when reconstituted PFL-AE is not isolated from the reconstitution reaction, or when the reconstituted enzyme is subjected to gel filtration with iron and sulfide in the gel filtration buffer. In both cases, “added iron” is present in the assay due to the presence of iron in the protein buffer. Furthermore, they report that when their reconstituted PFL-AE is subjected to Superdex 75 gel filtration in the absence of iron and sulfide in the buffer, ⱖ90% of the enzymatic activity is lost (for the assay done in the absence of added iron). Our current results show that purified native PFL-AE that has not been subjected to reconstitution, and therefore has no exogenous iron or sulfide in its buffer, exhibits catalytic activity in the absence of added iron. This result unequivocally demonstrates

302 cm ⫺1. The energies and relative intensities of the observed bands are very similar to those reported for [3Fe– 4S] ⫹ forms of ferredoxins (24 –26) and aconitase (27). Based on the cluster vibrational assignments determined by Johnson and co-workers using 34S isotopic labeling and normal mode analysis (24), we can tentatively assign the peaks at 270, 302, and 343 cm ⫺1 as primarily Fe–S bridging in nature, while the 378 cm ⫺1 and 417 cm ⫺1 peaks are primarily Fe–S terminal modes. Together, the UV–Vis, EPR, and Raman studies support the presence of [3Fe– 4S] ⫹ as the primary cluster form in as-isolated native PFL-AE. DISCUSSION Through the use of strictly anaerobic conditions during purification, we have succeeded for the first time in isolating PFL-AE with nearly a full complement of iron-sulfur cluster. The purified enzyme contains approximately 3 irons and 3 sulfides per protein molecule, and exhibits a specific activity of 31 U/mg in the absence of exogenous iron in the activity assay. This activity is approximately 75% of that observed in the presence of added iron, suggesting 75% occupancy of

FIG. 3. Resonance Raman spectrum of pyruvate formate-lyase activase. The enzyme is 0.39 mM in 50 mM Hepes/200 mM NaCl, pH 7.2. The data were collected using an experimental setup described previously. The sample was contained in a spinning cell in an anaerobic atmosphere, and was cooled to ⬍10°C during data collection. The laser power at the sample was 35 mW and the spectral slit width was 3 cm ⫺1. Total accumulation time was 1 h. The spectrum was corrected for contributions from buffer and baseline but was not smoothed.

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that we have isolated a functionally relevant form of PFL-AE, and furthermore that the only significant role for iron in previously reported PFL-AE assays is in cluster reconstitution. This result also highlights a difference between the native and reconstituted PFL-AE: while gel filtration on Superdex 75 resulted in ⱖ90% loss of enzymatic activity for reconstituted PFL-AE (20), we routinely use Superdex 75 gel filtration to isolate our native, catalytically active, PFL-AE. Reconstitution of PFL-AE, therefore, produces an enzymebound iron-sulfur cluster with properties distinct from those of the native enzyme. A combination of UV-visible, EPR, and resonance Raman spectroscopic studies have shown that the enzyme purified under strictly anaerobic conditions contains primarily a [3Fe– 4S] ⫹ cluster. By analogy to other enzymes in which a [3Fe– 4S] ⫹ cluster has been identified, it is expected that the catalytically active cluster of PFL-AE will be a [4Fe– 4S] cluster, and that this cluster is generated under the reducing conditions present in the assay. Such behavior has been observed previously with aconitase, in which the [3Fe– 4S] ⫹ cluster is converted to the active [4Fe– 4S] form by incubation under reducing conditions (28). The purification of PFL-AE containing primarily a [3Fe– 4S] ⫹ cluster implies a labile fourth iron site, and is consistent with the observation that only three cysteines have been implicated in cluster coordination (20). These three cysteines exist in a CX 3CX 2C motif that is common to all of the Fe-S/AdoMet enzymes for which a sequence has been determined (29 –34). The identification of an iron–sulfur cluster in PFL-AE places it among an unusual and emerging group of enzymes that utilize iron–sulfur clusters and AdoMet to initiate biologically important radical chemistry. This group of enzymes includes other activases that generate catalytically essential glycyl radicals (31,35,36), enzymes involved in cofactor biosynthesis (37– 40), an enzyme catalyzing a rearrangement reaction (41), and an enzyme involved in repair of DNA damage (42). Despite the diverse reactions catalyzed by these enzymes, the accumulating evidence points to a common mechanism involving the generation of an intermediate 5⬘-dAdo radical. How such a radical intermediate can be generated by the interaction of AdoMet with an iron-sulfur cluster remains a central, unresolved question regarding the Fe–S/AdoMet enzymes. ACKNOWLEDGMENTS This work was supported by grants from the NIH (GM54608 to J.B.B.), Research Corporation (CC4057 to J.B.B.), and the NSF-REU program (M.K.), and by startup funds provided by Amherst College and Michigan State University. A portion of this work was conducted at Amherst College. The authors thank John Kozarich for supplying pMG-AE and pKK-PFL and for useful advice in the early stages of this project. The authors also thank Adrian Goldman for helpful discussions regarding PFL expression.

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