COMMUNICATION
One Protein, Two Enzymes*

Yong Dai, Pieter C. Wensink, and Robert H. AbelesDagger

From the Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02453-2728

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

Two enzymes, designated, E-2 and E-2', catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2', overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2' are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni2+ or Co2+ to the apo-protein yields E-2 activity. E-2' activity is obtained when Fe2+ is added. Production in intact E. coli of E-2 and E-2' depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

S-Methylthioadenosine, a metabolite derived from methionine (1, 2), is a strong inhibitor of polyamine biosynthesis and transmethylation reactions (2, 3). Therefore its concentration in biological systems must be tightly controlled. Control is achieved through a ubiquitous metabolic pathway called the methionine salvage pathway, which catalyzes conversion of the 5-methylthio-D-ribose moiety of S-methylthioadenosine to methionine (4-7).

In Klebsiela pneumoniae where all intermediates of the pathway have been identified (4-7), metabolism of an aci-reductone is a branch point in the pathway (Fig. 1) (8-10). This molecule can undergo either a 1,2-oxygenlytic reaction to yield the alpha -keto acid precursor of methionine (Reaction 1) or a 1,3-oxygenlytic reaction to yield CO, formate, and methylthiopropionic acid (Reaction 2). The purpose of the off-pathway transformation of the aci-reductone (Reaction 2) is unclear. CO may simply be an easily cleared byproduct. Recent experiments in mammals, however, have established CO as a diffusible neurotransmitter, acting in a similar manner to that of nitric oxide (11, 12). CO may also play a role as a messenger in bacteria.


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Fig. 1.   Reactions of aci-reductones. III, an aci-reductone intermediate in the methionine salvage pathway, is a naturally occurring substrate for E-2 and E-2'. IIIa, a desthio analog of III, was found to be an alternate substrate (10).

In a previous study we purified E-2, which catalyzes Reaction 2, to near homogeneity from K. pneumoniae (8). To study the structure and catalytic action of E-2, we decided to clone and overproduce the enzyme in Escherichia coli. To our surprise, E-2' which catalyzes Reaction 1, the other branch of the pathway, is overproduced in the same E. coli cells. In this report we describe the purification and characterization of both enzymes and demonstrate that their distinct catalytic and chromatographic properties result from binding different metals.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Expression and Purification of E-2 and E-2'-- The E-2 gene was inserted at the initiator methionine codon of pET11a (13), a T7 RNA polymerase expression system. E. coli strain BL21, transformed with pET11a-E2, was grown in M9 medium (14) containing 100 mg/liter ampicillin at 37 °C. When the absorbance at 600 nm reached 0.35, 0.3 mM isopropyl-D-thiogalactoside was added to induce E-2 expression at 25 °C. The cells were incubated for an additional 12 h at 25 °C, harvested by centrifugation, and stored frozen at -70 °C.

The purification scheme was developed from the procedure used for purifying native E-2 from K. pneumoniae (8). Frozen BL21 cells were thawed in four volumes (w/v) of 100 mM phosphate buffer (KPi) (pH 7.5). All subsequent steps for purification were at 4° °C, unless otherwise noted. The suspension was sonicated 6 × 45 s with a Fisher sonic Dismembrator model 300 at 75% of maximal output. The sonicate was centrifuged at 30,000 × g for 30 min, and the precipitate was discarded. To the supernatant fluid was added 15 mg protamine sulfate/g of bacterial paste. After 30 min the suspension was centrifuged at 30,000 × g for 20 min, and ammonium sulfate (13.7 g/100 ml, 25% saturation) was added to the supernatant fluid. The suspension was centrifuged at 20,000 × g for 20 min. The supernatant fluid was brought to 70% saturation with ammonium sulfate (27.6 g/100 ml) and then centrifuged at 20,000 × g for 20 min. The precipitate was taken up in 30 mM KPi (pH 7.5) and applied to a Sephadex G-75 column (4.8 × 90 cm). The column was equilibrated and eluted with the same buffer. Fractions (20 ml/tube) were collected. The active fractions (35-45) were pooled and then concentrated by ultrafiltration using an Amicon PM10 membrane to 20 ml. In this step enzymatic activity was determined by measuring the loss in absorption of 0.1 mM IIIa (a desthio analog of III) in 50 mM KPi (pH 7.5) at 305 nm. The concentrate was chromatographed on a Amersham Pharmacia Biotech FPLC apparatus at 25 °C. Batches of 100 mg of protein were applied to a MonoQ 10/10 column that was pre-equilibrated with buffer A (20 mM Tris·HCl, pH 7.5). At a flow rate of 5 ml/min, the column was eluted with 20 ml of buffer A and then 80 ml of a linear gradient with buffer B (1 M NaCl, 20 mM Tris·HCl, pH 7.5) increasing from 0 to 15%, followed by 300 ml of a linear gradient with buffer B increasing from 15 to 30%. From this step on, the activities for oxidation of IIIa were monitored not only by observing consumption of IIIa and O2 but also by identifying the products. E-2 activity eluted at 115-130 ml. E-2' activity eluted at 150-190 ml. Batches of 5 mg of protein in buffer C (1.2 M (NH4)2SO4, 20 mM Tris·HCl, pH 7.5) were applied to a phenyl-Superose 5/5 column (Amersham Pharmacia Biotech) pre-equilibrated with buffer C. At a flow rate of 0.4 ml/min, the column was eluted with 2 ml of buffer C and then 4 ml of a linear gradient with buffer A increasing from 0 to 40%, and then 30 ml of a linear gradient with buffer A increasing from 40 to 100%. E-2 activity eluted at 8-11 ml. E-2' activity eluted at 16-24 ml.

Product Analyses-- The products derived from IIIa in Reactions 1 and 2 are formate, 2-oxopentanoic acid, butyrate, and CO. CO was quantitated by its reaction with deoxyhemoglobin (15). The other products were identified by 1H and 13C NMR spectroscopy. Formate and 2-oxopentanoic acid were quantitated by the formate and alpha -keto acid assays (10). Butyrate was quantitated by high pressure liquid chromatography using an organic acid column (9).

Removal and Replacement of the Metal Ion in E-2 and E-2'-- Apo-enzyme was prepared by dialysis of enzymes (10 mg/ml) against 30 mM EDTA in buffer A. The apo-enzyme could also be prepared from E-2 or E-2' by denaturing in 6 M guanidine-HCl in the presence of 5 mM EDTA in buffer A and then renaturing by removing guanidine-HCl through dialysis. 2-3-fold molar excess of metal over protein was then added to the apo-enzyme in buffer A, and the mixture was incubated for 10 min at 4 °C, followed by dialysis against buffer A to remove the excess metal. All experiments in which Fe2+ was used were carried out under anaerobic conditions.

Mass, Sequence, and Metal Analyses-- Electrospray mass spectra were obtained at Harvard University, Department of Chemistry. Metal contents of enzymes were determined by instrumental neutron activation analysis at MIT, Nuclear Reactor Laboratory. N-terminal sequence analysis of enzymes were performed in Applied Biosystems 477A at Tufts University, Department of Physiology.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Two Enzymes Are Produced from the Same Gene-- Based on the N-terminal sequence of E-2 purified from K. pneumoniae, we identified the E-2 gene in pDiox-2, a plasmid constructed by Yocum and co-workers (16) and shown by them to contain several other genes of the methionine salvage pathway of Klebsiela oxytoce, a derivative of K. pneumoniae (16). We sequenced the entire E-2 gene (GenBankTM accession number AF102514), which encodes a 179-amino acid polypeptide. This E-2 gene was overexpressed in E. coli. The overproduced enzyme was then purified. Surprisingly, when the partially purified, overproduced enzyme was chromatographed on a MonoQ or Phenylsuperose column, and two protein bands were detected (Figs. 2 and 3). Re-chromatography of each band on the same column yielded a single band. The protein from one band catalyzes Reaction 1, and the protein from the other band catalyzes Reaction 2 (Fig. 1). The proteins, designated E-2' and E-2, respectively, were purified to >= 95% homogeneity as measured by SDS-polyacrylamide gels (Fig. 4). The turnover numbers of E-2 and E-2' were 500 and 210 s-1, respectively. The overproduced E-2 is indistinguishable from the native E-2 from K. pneumoniae in catalytic activity measured by formate and CO formation and chromatographic properties.


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Fig. 2.   MonoQ chromatography of E-2 and E-2'. Partially purified enzyme was loaded onto MonoQ 10/10 column (Amersham Pharmacia Biotech) and eluted with buffers A and B as described under "Experimental Procedures."


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Fig. 3.   Phenylsuperose chromatography of E-2 and E-2'. The concentrated E-2 (panel A) or E-2' (panel B) fractions from MonoQ Chromatography (Fig. 2) were loaded onto Phenylsuperose 5/5 column (Amersham Pharmacia Biotech) and eluted with buffer C and buffer A as described under "Experimental Procedures."


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Fig. 4.   SDS-polyacrylamide gel electrophoresis of the purification of overexpressed E-2 and E-2'. Lane 1, crude extract from induced cells. Lane 2, 25-70% saturation of ammonium sulfate cut. Lane 3, the combined E-2/E-2' peak from Sephadex G-75. Lane 4, E-2 fraction from MonoQ column. Lane 5, E-2' fraction from MonoQ column. Lane 6, E-2 fraction from phenyl-Superose column. Lane 7, E-2' fraction from phenyl-Superose column. Lane 8, native E-2 from K. pneumoniae. Lane 9, molecular weight marker (Amersham Pharmacia Biotech catalog number 17-0446-01).

As expected, the two proteins encoded by the same gene are highly similar. The molecular weights of E-2 and E-2' were determined by mass spectrometry and found to be 20,252 ± 20 and 20,238 ± 20 Da, in close agreement with the 20,184 Da predicted from the open reading frame of the gene. The amino acid sequences (5 amino acids) from their N termini were found to be identical. As shown in Fig. 2 and 3, E-2 and E-2' can be separated on MonoQ and Phenylsuperose columns, but they co-chromatograph as monomers on a Sephadex G-75 column. These results suggest that E-2 and E-2' are of the same size, probably have the same amino acid composition, but differ in surface charge distribution, hence the separation on the MonoQ column.

The Two Enzymes Can Be Interconverted by Changing the Bound Metals-- We considered the possibility that the proteins differ in metal ion content. To examine this possibility, apo-enzyme with undetectable catalytic activity was produced from both E2 and E2' by prolonged dialysis against high concentrations of EDTA or by denaturing in guanidine and then renaturing. Addition of Ni2+ or Co2+ to the apo-enzyme, whether derived from E-2 or E-2' or prepared by either method, yielded (>90%) E-2 activity. Addition of Fe2+ to any of these apo-enzyme preparations yielded (>90%) E-2' activity. Thus, the difference in catalytic activity of E-2 and E-2' is largely, possibly entirely, due to the metal component of the enzymes. Further, the interconvertability of E-2 and E-2' provides additional evidence for the identity between the amino acid sequences of E-2 and E-2'.

Affinity of the Apo-enzyme for Ni2+ and Fe2+-- Both E-2 and E-2' retain full activities after 4 days of dialysis against 0.1 mM EDTA, indicating very low dissociation rates for Ni2+ and Fe2+. However, the rate of dissociation of Fe2+ substantially increased (t1/2 = 40 h) when E-2' was dialyzed against 5 mM Ni2+, indicating that the nickel ion participates in Fe2+ dissociation. In contrast, dialysis of E-2 against 5 mM Fe2+ produced no measurable increase in Ni2+ dissociation, suggesting a lower affinity for Fe2+ than Ni2+. This suggestion is supported by the observation that simultaneous addition of three equivalents of both Ni2+ and Fe2+ produced >80% E-2 within 1 min. Although these observations bear further investigation, they indicate a substantially higher affinity for Ni2+ than Fe2+.

We also investigated the effect of metals on the production of E-2 and E-2' in E. coli. The cells were grown and expression was induced in M9 minimum medium (14). The relative amount of E-2:E-2' was approximately 1:3. When expression was induced in the same medium, enriched by addition of 1 µM Ni2+ or Co2+, the relative amount of E-2 to E-2' increased to 10:1. Iron-enriched M9 medium did not change the relative amount of E-2 to E-2'. These results demonstrated that the production of E-2 and E-2' is strongly dependent on the availability of metals.

The metal content of the E-2 and E-2' overexpressed in Ni2+ and iron-enriched medium, respectively, was determined by instrumental neutron activation analysis and found to be 1.1 ± 0.4 mole atoms of Ni2+/mole E-2 and 0.9 ± 0.2 mole atoms of Fe2+/mole E-2'. When overexpressed in unenriched medium, a mole of E-2 contains 0.8 ± 0.3 mole atoms of Ni2+ and a mole of E-2' contains 0.6 ± 0.3 mole atoms of Fe2+.

One Protein Yields Two Enzymes with Different Catalytic Activities-- The enzymes, E-2 and E-2', catalyze different reactions (Reaction 1 and 2) but have the same protein component. They have similar molecular weights as determined by mass spectrometry and Sephadex chromatography. Their N-terminal amino acid sequences (5 amino acids) are identical. Most striking is the demonstration that the two enzymes can be interconverted. The distinction between the two enzymes appears to derive entirely from the metal component. For example, the Ni2+ enzyme catalyzes Reaction 2 but not Reaction 1.

The metal-dependent interconversion between E2 and E2' appears to have no precedent in the literature. Some metalloenzymes, such as carboxypeptidase, carbonic anhydrase, and alkaline phosphatase, have shown metal-dependent change in activity, but they still catalyze the same reactions (17-19). However, we are unaware of any previous reports of a metal-dependent change in the reaction products from the same substrate.

Plausible Catalytic Mechanisms for the Two Enzymes-- How can two nearly identical enzymes catalyze two different reactions? We considered the possibility that E-2 might make RCOOH and CO by further decarbonylating the alpha -ketoacid product of E-2', RCOCOOH. However, E-2 does not show any activity on RCOCOOH (kcat/Km <10-4 M-1 s-1), indicating that Reactions 1 and 2 are not sequential. Our previous isotope tracer studies of purified E-2 and E-2' activity in extracts and model experiments for CO production led us to propose a speculative mechanism (9) that could lead to formation of two different sets of products. An abbreviated version of the mechanism is shown in Fig. 5. The hydroperoxide radical or anion adds to C-2 or C-3. Addition to C-3 produces formate, CO, and butyrate. Addition to C-2 produces formate and 2-oxopentanoic acid. If this mechanism is correct, then it is likely that the metal ion affects the structure of the active site and thereby determines the point of addition of the hydroperoxide radical or anion and consequently the nature of the products.


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Fig. 5.   Postulated mechanism of product formation.


    ACKNOWLEDGEMENTS

We thank Patricia Murray, who helped in preparing the manuscript, and Dr. Theodore Alston for helpful discussion.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 2 R01 GM12633.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF102514.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Brandeis University, 415 South St., Waltham, MA 02453-2728. Tel.: 781-736-2310; Fax: 781-736-2349; E-mail: abeles{at}binah.cc.brandeis.edu.

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

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