1 Department of Pediatrics, University of Florida, Gainesville, FL 32611, USA
2 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA
Correspondence
Thomas A. Bobik
bobik{at}iastate.edu
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ABSTRACT |
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INTRODUCTION |
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The pathway of 1,2-PD degradation begins with the conversion of 1,2-PD to propionaldehyde by AdoCbl-dependent diol dehydratase (Bobik et al., 1997; Obradors et al., 1988
; Toraya et al., 1979
). Subsequently, propionaldehyde is converted to propanol and propionate by alcohol dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacylase and propionate kinase (Obradors et al., 1988
; Toraya et al., 1979
). This pathway generates one ATP, an electron sink for the regeneration of NAD and an intermediate (propionyl-CoA) that can serve as a carbon and energy source via the methylcitrate pathway (Horswill & Escalante-Semerena, 1997
).
Although the pathway of 1,2-PD degradation appears quite straightforward, the catabolism of this small molecule is in fact a very elaborate process (Bobik et al., 1999; Havemann et al., 2002
; Havemann & Bobik, 2003
). Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al., 1999
; Havemann et al., 2002
; Havemann & Bobik, 2003
). These polyhedra are 100150 nm across, consist of a proteinaceous shell and interior and are composed of at least 15 different polypeptides, including four enzymes essential for 1,2-PD degradation (Havemann & Bobik, 2003
). While our understanding of these unusual structures is still relatively limited, studies conducted so far indicate that their role is to mitigate the toxicity of propionaldehyde, an obligatory intermediate of the 1,2-PD catabolic pathway (Havemann et al., 2002
; Havemann & Bobik, 2003
; Leal et al., 2003a
).
Adding to the complexity of 1,2-PD degradation is the fact that this process requires systems for the acquisition and maintenance of AdoCbl. S. enterica can obtain AdoCbl by de novo synthesis and by uptake from the environment (Roth et al., 1996). In addition, S. enterica can transport corrinoid precursors such as hydroxycobalamin (HOCbl) and cyanocobalamin (CNCbl; vitamin B12) and convert these compounds to AdoCbl (Roth et al., 1996
). The de novo synthesis of AdoCbl occurs only under anoxic conditions, but the use of exogenous AdoCbl or complex precursors proceeds both aerobically and anaerobically. The use of corrinoid precursors requires addition of the appropriate upper ligand to the central cobalt atom of the corrin ring, which requires several enzymic steps (Huennekens et al., 1982
; Vitols et al., 1965
; Weissbach et al., 1961
) (Fig. 1
). First, CNCbl is converted to HOCbl by cobalamin
-ligand transferase (Brady & Barker, 1961
; Pezacka et al., 1990
; Pezacka, 1993
; Watanabe et al., 1987
; Weissbach et al., 1962
). Next, two successive one-electron reductions of the central cobalt atom of HOCbl lead to the formation of cob(II)alamin and cob(I)alamin (Brady & Barker, 1961
; Brady et al., 1962
; Fonseca & Escalante-Semerena, 2000
, 2001
; Fujii & Huennekens, 1974
; Fujii et al., 1977
; Pezacka, 1993
; Walker et al., 1969
; Watanabe et al., 1987
, 1993
, 1996
; Weissbach et al., 1961
). These reactions are catalysed by cob(III)alamin reductase and cob(II)alamin reductase, respectively. Lastly, ATP : cob(I)alamin adenosyltransferase catalyses the transfer of a 5'-deoxyadenosyl group from ATP to cob(I)alamin, forming AdoCbl (Brady et al., 1962
; Debussche et al., 1991
; Dobson et al., 2002
; Johnson et al., 2001
; Leal et al., 2003b
; Peterkofsky et al., 1961
; Suh & Escalante-Semerena, 1995
; Vitols et al., 1965
). Interestingly, this pathway has a dual function. In addition to its role in the assimilation of exogenous corrinoid compounds, it is also needed for recycling HOCbl generated endogenously from the breakdown of AdoCbl, which is unstable during catalysis (Toraya & Mori, 1999
; Toraya, 2000
).
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Earlier studies of 1,2-PD degradation have indicated that the pdu operon includes genes for the conversion of CNCbl to AdoCbl, but so far only the pduO gene has been shown to be involved in this process (Johnson et al., 2001). Recent bioinformatic analyses indicated that the PduS protein has ironsulfur and NAD-binding motifs, suggesting that this enzyme might be involved in cobalamin reduction (unpublished data). In this report, we present in vitro evidence that the PduS enzyme is a bifunctional cobalamin reductase used for the conversion of CNCbl into AdoCbl.
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METHODS |
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Bacterial strains and media.
The bacterial strains used in this study were S. enterica serovar Typhimurium LT2 (formerly S. typhimurium LT2), Escherichia coli DH5 and E. coli BL21 DE3 RIL (Stratagene). LB (LuriaBertani) medium was the rich medium used (Miller, 1972
).
General molecular methods.
Agarose gel electrophoresis was performed as described by Maniatis et al. (1982). Plasmid DNA was purified by the alkaline lysis procedure (Maniatis et al., 1982
) or by using Qiagen products according to the manufacturer's instructions. Following restriction digestion or PCR amplification, DNA was purified using Qiagen PCR purification and gel extraction kits or by using phenol/chloroform extraction followed by ethanol precipitation (Maniatis et al., 1982
). Restriction digestions were carried out using standard protocols (Maniatis et al., 1982
). For ligation of DNA fragments, T4 DNA ligase was used according to the manufacturer's instructions. Electroporation was used for bacterial transformation. A Gene Pulser (Bio-Rad) was used according to the manufacturer's instructions and at the following settings: capacitance, 25 µF; capacitance extender, 250 µF; pulse controller, 200
; voltage, 2·5 kV. LB medium containing the appropriate antibiotic(s) was used to select for transformed cells and, prior to the analysis of transformants, pure cultures were prepared.
General protein methods.
PAGE was performed using Bio-Rad Redigels and Mini-protean II electrophoresis cells. PAGE was run at 200 V (constant voltage) for 45 min using a Bio-Rad Powerpac 300. Following gel electrophoresis, Coomassie brilliant blue R-250 was used to stain proteins. The protein concentration of solutions was determined using Bio-Rad protein assay reagent according to the manufacturer's instructions with BSA as the standard.
DNA sequencing and analysis.
DNA sequencing was carried out by the University of Florida Interdisciplinary Center for Biotechnology Research DNA Sequencing Core Facility using Applied Biosystems Inc. automated sequencing equipment (Perkin Elmer). The template for DNA sequencing was plasmid DNA purified using Qiagen tip 100 columns. BLAST software was used for sequence similarity searches (Altschul et al., 1990, 1997
).
Cloning the pduS gene for high-level expression.
PCR was used to amplify the pduS coding sequence for cloning into a modified T7 expression plasmid, pTA925 (Johnson et al., 2001). The Pfu polymerase was used because of its high fidelity of DNA replication. Template pMGS2 was used with primers 5'-GGAATTCAGATCTTATGAGCACCGCCATCAACAG-3' (forward) and 5'-GGAATTCAAGCTTTGGTTAACCTCTTACAACAGTG-3' (reverse). These primers introduced the BglII and HindIII restriction sites that were used to clone the pduS coding sequence into pTA925. Ligation mixtures were used to transform E. coli strain DH5
and transformants were selected by plating on LB agar supplemented with 25 µg kanamycin ml1. Plasmid DNA isolated from selected transformants was analysed by restriction digestion and DNA sequencing. One plasmid that contained a pduS insert with the expected DNA sequence (pAP253) was introduced into expression strain E. coli BL21 DE3 RIL by electroporation and the resulting strain (BE311) was used for production of recombinant PduS protein.
Growth of the PduS expression strain and preparation of cell extracts.
E. coli strain BE311 was grown in 500 ml LB kanamycin (25 µg ml1) broth incubated at 20 °C with shaking at 275 r.p.m. in a 1 litre baffled Erlenmeyer flask. Cells were grown to an OD600 of 0·60·8. Next, expression of the pduS gene was induced by the addition of IPTG to a concentration of 0·5 mM. Cells were incubated for an additional 812 h and harvested by centrifugation at 6690 g for 10 min using a Beckman JLA-10.500 rotor. Following centrifugation, all subsequent procedures were carried out using anaerobic procedures. Two grams of cells (wet weight) were resuspended in 3 ml of a solution containing 50 mM sodium phosphate pH 7, 50 mM KCl, 1 mM DTT and 0·24 mg of the protease inhibitor PefaBloc ml1 (Pentapharm). Cells were broken using a French pressure cell (SLM Aminco) at 20 000 p.s.i. Soluble proteins were separated from inclusion bodies by centrifugation of cell extracts at 31 000 g for 30 min using a Beckman JA-20 rotor. The supernatant obtained was the soluble cell extract used for further studies. The pellet was treated with Bacterial Protein Extraction Reagent II (Pierce Biotechnology) according to the manufacturer's instructions and under anoxic conditions to obtain the purified inclusion bodies used for further analyses. Control strain BE119 (E. coli BL21 DE3 RIL/pTA925-no insert) was grown in parallel with expression strain BE311 and cell extracts of this strain were similarly prepared.
Cob(III)alamin reductase assays.
Cob(III)alamin reductase assays were a modification of a previously described method (Watanabe et al., 1987). Assay mixtures contained 50 mM CHES/NaOH (pH 8·5), 1·6 mM KH2PO4, 2·8 mM MgCl2, 0·5 mM NADH and 100 µM HOCbl. The total assay volume was 2 ml and assays were carried out using strict anaerobic precautions. Inside an anoxic chamber (Coy Laboratory Products), assay components were dispensed into modified glass cuvettes, sealed with 13 mm grey butyl rubber stoppers and aluminium crimp seals, removed from the chamber and flushed with N2 for 30 s (Johnson et al., 2001
). Cuvettes were placed in a 37 °C water bath for 5 min and reactions were initiated by adding a source of enzyme or a particular assay component using the following procedures to minimize the introduction of oxygen. The assay component to be added was placed within a sealed serum vial and flushed with N2 for 2 min. Additions were made using a Hamilton syringe that had been flushed with anoxic water just prior to use. Reaction rates were determined by monitoring the decrease in absorbance at 525 nm and using
525=4·9 mM1 cm1 for calculations.
Linked cob(II)alamin reductase assays.
For the measurement of cob(II)alamin reductase activity, a linked assay with the PduO adenosyltransferase was used (Fig. 2). The basis of this assay is that the PduO enzyme converts cob(I)alamin to AdoCbl, which occurs with a concomitant increase in absorbance at 525 nm (
525=4·8 mM1 cm1). PduO is specific for cob(I)alamin and does not react with cob(II)alamin (Johnson et al., 2001
). Anaerobic precautions were used as described above for cob(III)alamin reductase assays. Reaction mixtures contained 50 mM CHES/NaOH (pH 8·5), 1·6 mM KH2PO4, 2·8 mM MgCl2, 100 µM cob(II)alamin, 0·4 mM ATP, 1 mM NADH, 120 µg purified PduO adenosyltransferase and a source of cob(II)alamin reductase. Assays were carried out at 37 °C and reactions were initiated by adding a source of enzyme or a particular assay component as indicated in the text. Cob(II)alamin was generated by exposing a 10 mM AdoCbl stock solution to a 150 W incandescent light at a distance of 20 cm for 1020 min under anaerobic conditions at room temperature. Purified PduO adenosyltransferase was obtained from recombinant E. coli strain BE118 (Johnson et al., 2001
; unpublished data).
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HPLC analysis of AdoCbl, CM-Cbl and HOCbl.
Reverse-phase HPLC was used to separate and quantify HOCbl, AdoCbl and CM-Cbl. A NovaPak C18 column (3·9x150 mm) equipped with a C18 Sentry guard column was used for the separation (Waters). Samples (200 µl) were loaded onto the column and eluted with 30 min linear gradient of 10 to 90 % methanol in 100 mM sodium acetate (pH 4·6) at a flow rate of 1 ml min1. The absorbance of effluent was monitored at 365 nm and analytes were quantified by comparison of peak areas to a standard curve. The CM-Cbl standard was prepared by incubating a solution of 200 mM Tris/HCl pH 8·0, 1·6 mM potassium phosphate, 2·8 mM MgCl2, 100 mM KCl, 100 µM cob(II)alamin, 1 mM DTT, 50 µM FAD and 0·4 mM iodoacetate at 37 °C for 1 h under strictly anaerobic conditions, which resulted in quantitative conversion of cob(II)alamin to CM-Cbl (data not shown).
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RESULTS |
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The PduS protein has cob(II)alamin reductase activity
Cell extracts from the PduS expression strain and the control strain were also assayed for the ability to reduce cob(II)alamin to cob(I)alamin [cob(II)alamin reductase activity]. The assay used was a linked assay, in which the cob(I)alamin formed was converted to AdoCbl by the PduO adenosyltransferase (Fig. 2). In assays that contained standard components, 100 µM cob(II)alamin, 0·4 mM ATP, 1 mM NADH, 120 µg purified PduO and 500 µg crude PduS extract, the rate of cob(I)alamin formation averaged 3·8±0·3 nmol min1 in three trials. No activity was detected in similar assays when the PduS extract was replaced with the control extract. For these assays, a kinetic excess of PduO was used (data not shown), and controls showed that omission of purified PduO, crude PduS, NADH, ATP or cob(II)alamin eliminated detectable cob(II)alamin reductase activity. In addition, three tests were used to verify that AdoCbl was the reaction product: (i) the UV-visible spectrum of completed reactions was characteristic of AdoCbl (Fig. 4
), (ii) the reaction product co-migrated with authentic AdoCbl by reverse-phase HPLC following co-injection (not shown) and (iii) the product was photolysed by a 30 min exposure to incandescent light with the formation of cob(II)alamin (not shown).
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To test whether cob(I)alamin was sequestered or released free in solution during the conversion of cob(II)alamin to AdoCbl by the PduSPduO system, a chemical trap for cob(I)alamin was used. Iodoacetate, which reacts rapidly and quantitatively with cob(I)alamin to form CM-Cbl (Fonseca & Escalante-Semerena, 2000, 2001
), was added to a PduSPduO linked cob(II)alamin reductase assay in a 250-fold molar excess compared with the PduO enzyme (400 µM compared with 1·6 µM). The reaction was allowed to proceed to completion (30 min at 37 °C). CM-Cbl and AdoCbl were then resolved and quantified by reverse-phase HPLC. Results showed that 84 % of the cob(I)alamin formed was converted to AdoCbl and only 16 % was converted to CM-Cbl, even though iodoacetate was present in a large molar excess compared with the PduO adenosyltransferase (Table 1
, row 1). This result indicated that cob(I)alamin was sequestered during the conversion of cob(II)alamin to AdoCbl by the PduS-PduO system. If cob(I)alamin had been released free in solution, the majority should have been converted to CM-Cbl by reaction with the iodoacetate, which was present in large excess compared with PduO.
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Moreover, controls showed that the difference in CM-Cbl formation between the chemical and enzymic systems could not be accounted for on the basis of differences in the kinetics of cob(I)alamin formation. When DTT plus FAD were used to generate free cob(I)alamin, the rate of cob(I)alamin production was similar to the rate observed for the PduSPduO system. (In the absence of iodoacetate, assays that contained DTT, FAD and purified PduO produced AdoCbl at a rate of 3·5 nmol min1, which was very close to the rate at which AdoCbl was produced in assays containing the PduS and PduO enzymes, 3·8 nmol min1).
It is also of note that iodoacetate had relatively little effect on the total amount of AdoCbl produced from cob(II)alamin by the PduS and PduO enzymes (Table 1, rows 1 and 3). In the absence and presence of iodoacetate, 128 and 114 nmol AdoCbl was produced, respectively. This indicates that the majority of the cob(I)alamin formed [during the conversion of cob(II)alamin to AdoCbl by the PduSPduO system] was sequestered.
PduS does not produce significant amounts of cob(I)alamin in the absence of the PduO protein
We also used iodoacetate to test whether the PduS protein can produce free cob(I)alamin in the absence of the PduO protein. Assay mixtures were prepared that were similar in composition to the PduSPduO linked assay described above except that PduO was omitted and iodoacetate was included. Iodoacetate reacts with cob(I)alamin to form CM-Cbl, with a concomitant increase in absorbance at 525 nm that provides a facile method for quantifying free cob(I)alamin that is slightly more sensitive than the linked assay with the PduO adenosyltransferase. In assays containing iodoacetate, no cob(I)alamin was detected over a 30 min period (Fig. 5). In contrast, cob(I)alamin was formed at a rate of 3·9 nmol min1 in the linked cob(II)alamin reductase assays that included the PduO enzyme. These findings show that PduS enzyme requires the presence of the PduO adenosyltransferase in order to reduce cob(II)alamin to cob(I)alamin at a detectable rate, which indicates a physical interaction between the PduS and PduO enzymes.
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DISCUSSION |
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Cobalamin reductases have been studied previously in S. enterica, Clostridium tetanomorphum, Propionibacterium shermanii, Euglena gracilis, rat, rabbit and human (Brady & Barker, 1961; Brady et al., 1962
; Fonseca & Escalante-Semerena, 2000
, 2001
; Fujii & Huennekens, 1974
; Fujii et al., 1977
; Pezacka, 1993
; Walker et al., 1969
; Watanabe et al., 1987
, 1993
, 1996
; Weissbach et al., 1961
; Wilson et al., 1999
). Of the enzymes that have been investigated, the corresponding gene is known in five cases. In S. enterica, the Fre protein was reported to catalyse the production of reduced flavin nucleotides which, in turn, reduce cob(III)alamin chemically to cob(II)alamin (Fonseca & Escalante-Semerena, 2000
). The E. coli FldA protein, the human methionine synthase reductase (MSR) and the human novel reductase 1 (NR1) were shown to reduce cob(II)alamin to cob(I)alamin for the reductive activation of methionine synthase (Fujii et al., 1977
; Olteanu & Banerjee, 2003
; Paine et al., 2000
; Wilson et al., 1999
). In addition, the FldA protein in combination with the Fpr protein was reported to reduce cob(III)alamin to cob(I)alamin for AdoCbl production by the CobA adenosyltransferase (Fonseca & Escalante-Semerena, 2001
).
The PduS protein is unrelated in amino acid sequence to the Fre, Fpr, FldA, MSR and NR1 proteins. Hence, PduS represents a new class of enzyme with cobalamin reductase activity. Analyses using BLASTP software showed that GenBank currently contains 52 proteins related in sequence to PduS (Expect value 7x1010 or lower) (Altschul et al., 1990, 1997
). It is likely that the PduS homologues found in Salmonella, Lactobacillus, Listeria and Klebsiella have conserved functions, since their encoding genes are located proximal to genes involved in AdoCbl-dependent 1,2-PD degradation (Bork et al., 1998
; Overbeek et al., 1999
). However, PduS also has homology to the RnfC subunit of NADH : ubiquinone oxidoreductase, and many of the PduS homologues currently in GenBank are arranged with genes predicted to encode this enzyme (rnfABCDGE). These enzymes probably have functions divergent from PduS, but an intriguing possibility is that they have roles in both electron transport and cobalamin reduction. Also of interest is the fact that PduS has significant similarity to a single human flavoprotein (GI:20149568; Expect value=8x106), but not to other human electron transport oxidoreductases. This finding could be helpful to studies of cobalamin reduction in humans, which has relevance to several disease states including hyperhomocysteinuria, methylmalonic aciduria, cancer and heart disease (Ames, 2001
; Rosenblatt & Fenton, 1999
).
In this report, we also conducted studies to determine whether cob(I)alamin was sequestered or released free in solution during the conversion of cob(II)alamin to AdoCbl by an in vitro system that contained the PduS cobalamin reductase and the PduO adenosyltransferase. Experiments in which iodoacetate was used as a chemical trap indicated that cob(I)alamin was sequestered during the conversion of cob(II)alamin to AdoCbl by the PduSPduO system. This is likely to be physiologically important. Cob(I)alamin is one of the strongest nucleophiles that exists in aqueous solution and an extremely strong reductant (Eo'=0·61 V) (Banerjee et al., 1990; Schrauzer et al., 1968
; Schrauzer & Deutsch, 1969
). It rapidly reduces protons to H2 gas at pH 7 and is instantaneously oxidized by air (Schneider, 1987a
, b
). Hence, sequestration of cob(I)alamin would prevent a futile cycle in which cob(II)alamin reduction is followed by the non-specific oxidation of cob(I)alamin due to its high reactivity. Thus, the finding that cob(I)alamin is sequestered suggests a specific and physiologically important interaction between the PduS and PduO enzymes.
Earlier studies have shown that the PduO adenosyltransferase is a component of the polyhedral bodies involved in 1,2-PD degradation by S. enterica (Havemann & Bobik, 2003). Results presented here indicate that PduS interacts physically with PduO; hence, the PduS protein should also be associated with these polyhedra. Recently, the polyhedral bodies of 1,2-PD degradation were purified and found to consist of at least 15 different polypeptides (Havemann & Bobik, 2003
). PduS was not among the proteins identified, but it could have been missed if it was a minor component. Alternatively, PduS could associate with the outer surface of the polyhedral bodies, in which case it might have been displaced by the detergent treatment used for polyhedral body purification.
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ACKNOWLEDGEMENTS |
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Received 5 November 2004;
revised 17 January 2005;
accepted 18 January 2005.
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