Programa de Ingeniería Metabólica, Centro de Investigación sobre Fijación de Nitrógeno, Universidad National Autónoma de México, A. P. 565-A, Cuernavaca, Morelos, Mexico
Correspondence
Michael F. Dunn
mike{at}cifn.unam.mx
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ABSTRACT |
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INTRODUCTION |
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The genome sequences of Sinorhizobium meliloti, Mesorhizobium loti (Dunn et al., 2002a) and Bradyrhizobium japonicum (Kaneko et al., 2002
) each contain homologues which potentially encode the four subunits of a heterotetrameric ACC similar to that of E. coli (Cronan & Waldrop, 2002
). Of particular significance to the studies presented here, these genomes also encode a number of
and
subunits which appear to compose two or more heteromeric acyl-CoA carboxylases in each organism.
Pyruvate carboxylase (PYC) fulfils an anaplerotic function in producing oxaloacetate from pyruvate and is the only biotin-dependent enzyme in rhizobia which has been extensively characterized (Dunn et al., 1996, 1997
, 2001
, 2002
). ACC and/or PCC activities have been reported in several rhizobia (DeHertogh et al., 1964
; Dunn et al., 2002
; Encarnación et al., 1995
; Lowe & Evans, 1962
) and could be catalysed in each species by distinct ACCs or PCCs and/or by a single acyl-CoA carboxylase. Western blot analysis of Rhizobium etli extracts revealed two biotin-containing proteins in addition to PYC (Dunn et al., 1996
). Based on their apparent molecular masses these proteins were hypothesized to be the
subunit of an acyl-CoA carboxylase and the AccB subunit of an ACC (Dunn et al., 1996
, 2002a
). Because our laboratory is studying the biosynthesis and utilization of biotin in R. etli, we wanted to identify and characterize the enzyme or enzymes responsible for catalysing the ACC and PCC reactions in this organism. We show here that R. etli produces an acyl-CoA carboxylase able to catalyse both the ACC and PCC reactions, but having a distinct kinetic preference for propionyl-CoA as substrate. Genome sequence analysis and enzyme activity assays suggest a universal distribution of the acyl-CoA carboxylase among rhizobia.
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METHODS |
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Purification of the R. etli acyl-CoA carboxylase.
Strain 12-53 was grown to early stationary phase in PY rich medium and cells from 6 l of culture were harvested by centrifugation at 9800 g and washed twice in 25 mM Tris/HCl (pH 7·0). This and all further operations were performed at 4 °C or on ice. The cell pellet was resuspended in 3 vols cell lysis buffer (CLB) [50 mM Tris/HCl (pH 7·0), 50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10 % (v/v) glycerol] and broken by three passages through a chilled French pressure cell at 20 000 p.s.i. The crude cell extract was obtained following centrifugation at 27 200 g for 20 min. Acyl-CoA carboxylase activity in the extract and in fractions obtained during purification were measured using the PCC assay. Proteins precipitating from the crude cell extract in the 3055 % of saturation range with ammonium sulfate were obtained by the gradual addition of the solid salt followed by a 20 min equilibration on ice without stirring and centrifugation at 10 000 g for 10 min.
The ammonium sulfate pellet was redissolved in AEC A buffer [25 mM Tris/HCl (pH 8·0), 5 mM MgCl2, 0·1 mM EDTA, 1 mM DTT, 2 % (v/v) glycerol] containing 200 mM KCl and dialysed against the same buffer. The sample was loaded onto a DEAE MacroPrep (Bio-Rad) column (5 cm diam.x5 cm length, flow rate 4 ml min-1, 10 ml per fraction) equilibrated in AEC A buffer containing 200 mM KCl and the column was washed in 300 ml of this buffer. This was followed by a 500 ml linear gradient of 200700 mM KCl in AEC A buffer, during which the acyl-CoA carboxylase activity eluted near the middle of the gradient. Active fractions were pooled and concentrated on a PLTK ultrafiltration membrane (Millipore) and loaded onto a ToyoPearl HW55 F (ToyoHaas) gel permeation column (2·5 cm diam.x95 cm length) equilibrated in GPC buffer [50 mM Tris/HCl (pH 7·0), 175 mM KCl, 5 mM MgCl2, 0·1 mM EDTA; 1 mM DTT, 2 % (v/v) glycerol] and eluted at 0·5 ml min-1, collecting 5 ml fractions. The active fractions were pooled and proteins were precipitated by the addition of ammonium sulfate to 80 % of saturation as described above.
The protein pellet obtained following centrifugation was redissolved in a small volume of HIC A buffer [100 mM sodium phosphate (pH 6·8), 4 M NaCl, 1 mM DTT, 2 % (v/v) glycerol], dialysed and applied to a 5 ml methyl HIC Econo-Pac cartridge (Bio-Rad) equilibrated in the same buffer at a flow rate of 1·5 ml min-1, collecting 1·5 ml per fraction. Following the elution of non-binding proteins with 15 ml HIC A buffer, the column was eluted with a 50 ml linear gradient terminating in 100 % HIC B buffer (HIC A buffer without NaCl). Fractions containing acyl-CoA carboxylase activity eluted mid-gradient. Active fractions were combined and buffer exchanged into AEC A buffer by ultrafiltration as described above. The sample (5 ml) was loaded onto a BioScale Q2 anion exchange column (Bio-Rad) and eluted with 6 ml AEC A buffer followed by a 20 ml linear gradient of 0400 mM KCl. The two fractions containing the highest activity were combined, made to 50 % (v/v) with glycerol and stored at -20 °C. This preparation was stable for at least 3 months.
Kinetic determinations.
Before use, the purified acyl-CoA carboxylase was dialysed against two changes (45 min each) of a 1000-fold excess of 25 mM Tris/HCl (pH 8·0) containing 1 mM DTT. Substrate Km and Vmax values were obtained from double-reciprocal plots. Stock solutions of potential water-soluble metabolic effectors were prepared in 50 mM Tris/HCl (pH 7·0). Fatty acid stock solutions were dissolved in 4 % (w/v) Brij 58 and added to the reaction mixture so as to have a final concentration of 0·4 % detergent in all reactions: control reactions contained this concentration of detergent without fatty acids. Salts were dissolved in water. The concentrations of salts or metabolic effectors giving 50 % inhibition or 50 % activation are reported as I[50] and S[50] values, respectively. All assays were done in triplicate unless stated otherwise.
SDS-PAGE and detection of biotin-containing proteins.
Electrophoresis, transblotting and detection of biotinylated proteins on Western blots was performed as described previously (Dunn et al., 1996).
Analytical gel permeation chromatography.
A BioSelect SEC400-5 gel column (Bio-Rad) equilibrated in GPC buffer (flow rate 0·5 ml min-1) was calibrated with 25 µl Bio-Rad Gel Filtration Standard, giving the following elution times: thyroglobulin (670 kDa), 19·67 min; -globulin (158 kDa), 21·83 min; ovalbumin (44 kDa), 24·17 min; myoglobin (12 kDa), 26·67 min. To determine the native molecular mass of the purified acyl-CoA carboxylase, a 25 µl sample of the enzyme [concentrated 10-fold and exchanged into GPC buffer on an Ultra-free MC ultrafiltration spin column (Sigma); total protein 1·1 µg] was injected onto the column under the same conditions used for calibration and 0·25 ml fractions were collected. PCC activity was determined in 10 µl aliquots of the fractions.
Analysis of reaction products by thin layer chromatography.
Radioassay reaction mixtures containing purified acyl-CoA carboxylase and propionyl- or acetyl-CoA as substrate were incubated for 16 h at 30 °C. Reactions were stopped by adding 3 vols methanol and the mixtures were evaporated to dryness under an air stream. The residues were redissolved in methanol and material equivalent to 15 µl of the original reaction was spotted onto a Silica gel 60 F254 thin layer chromatography plate (EM Science). Ten microgram quantities of authentic standards of acyl-CoAs, dissolved in methanol, were also spotted and the plate was developed in 1-butanol/acetic acid/water (12 : 3 : 5; Mohamed, 2000). Acyl-CoAs were visualized under UV light and 14C-labelled products were detected by autoradiography in a 6-day exposure with Kodak X-Omat K film.
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RESULTS AND DISCUSSION |
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ACC and PCC activities were present at wild-type levels in R. etli CFNX218 (Table 2), which is cured of all indigenous plasmids except for a portion of plasmid pe (Brom et al., 2000
). In addition, the biotin-containing protein corresponding to the acyl-CoA carboxylase
subunit (see below) was present in CFNX218. This indicates that the gene products required for acyl-CoA carboxylase activity are encoded on the chromosome in R. etli, in contrast to their plasmid location in S. meliloti (Charles & Aneja, 1999
).
The effect of culture carbon source and biotin supplementation on ACC and PCC activities in R. etli CE3 were determined. In unsupplemented minimal medium, activities of ACC and PCC were several-fold higher with pyruvate relative to succinate as sole carbon source. Supplementation of MM-pyruvate with biotin increased both ACC and PCC activities approximately twofold, while supplementation of MM-succinate resulted in a 10-fold increase in these activities. Similar biotin- and carbon-source-dependent effects on activity also occur with the R. etli PYC (Dunn et al., 1996, 1997
, 2002
), reinforcing the physiological data indicating the profound influence of growth conditions on the ability of R. etli to produce biotin de novo (Encarnación et al., 1995
). Neither the PCC/ACC ratios nor the absolute activities were significantly affected in biotin-supplemented MM or PY rich medium as a function of culture growth phase (results not shown).
Purification of the R. etli acyl-CoA carboxylase
In anion exchange and gel permeation chromatography experiments with R. etli strains CE3 and 12-53, both PCC and ACC activities always co-eluted (results not shown). To determine if the ACC and PCC activities present in R. etli cell extracts (Table 2) derived from a single acyl-CoA carboxylase, we purified the enzyme catalysing the PCC reaction from strain 12-53 (Table 2
). This strain lacks PYC and was initially chosen for purification work because this characteristic would be useful in exploiting avidin affinity chromatography for the selective purification of the acyl-CoA carboxylase. However, despite extensive trials with different monomeric avidin matrices under a variety of experimental conditions, we were unable to obtain significant binding of the enzyme to the column.
SDS-PAGE analysis of the enzyme purified as outlined in Table 3 showed two major protein bands of 74 and 45 kDa along with several proteins present in much lower amounts (Fig. 1
a). Subsequent blotting and detection with streptavidin-HRP conjugate showed that the 74 kDa band contained biotin (Fig. 1b
). We infer that the 74 kDa biotin-containing protein represents the
subunit of the acyl-CoA carboxylase and the 45 kDa protein corresponds to the
subunit of the enzyme. The purified acyl-CoA carboxylase eluted from a BioSelect SEC400 gel column with an elution time (19·75 min) corresponding to a protein with a molecular mass of 620 000 Da (Fig. 1c
). The subunit composition of prokaryotic acyl-CoA carboxylases has been determined only in Myxococcus xanthus (Kimura et al., 1998
) and Mycobacterium smegmatis (Haase et al., 1982
), and both are
6
6 enzymes. The experimentally determined molecular mass of the native R. etli acyl-CoA carboxylase fits fairly well with that calculated for an
6
6 structure (714 000 Da).
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A comparison of the apparent Km and Vmax values for the acyl-CoA carboxylase with acetyl-CoA, butryl-CoA or propionyl-CoA as substrates is shown in Table 4. The specificity constant, defined as Vmax/Km (Diacovich et al., 2002
), indicates the strong kinetic preference of the enzyme for propionyl-CoA as substrate. This is consistent with the observed high PCC/ACC ratios in cell extracts. Activity in reactions containing both propionyl-CoA (0·5 mM) and acetyl-CoA (0·5, 2 and 5 mM) decreased with increasing acetyl-CoA concentrations (reaching 38 % inhibition of activity at 5 mM, in comparison to control reactions lacking acetyl-CoA). These results are consistent with the acyl-CoA carboxylase having a single binding site for both substrates. Analysis of the reaction products of the purified enzyme by thin-layer chromatography demonstrated the formation of 14C-labelled products co-migrating with authentic malonyl-CoA and methyl-malonyl-CoA in reactions of the enzyme provided with acetyl-CoA and propionyl-CoA, respectively, as substrates (not shown).
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In reactions without added KCl, the PCC and ACC reactions of the purified acyl-CoA carboxylase have only 6 and 23 %, respectively, of the activity obtained in the standard assays (containing 40 mM KCl; Fig. 2 and results not shown). KCl gave S[50] values, relative to assays without salt, of 1·0 and 2·2 mM for the ACC and PCC reactions, respectively. In assays of PCC activity of the acyl-CoA carboxylase,
[as NH4Cl or (NH4)2SO4] or CsCl were as effective as KCl in stimulating the enzyme, while NaCl and LiCl were completely ineffective (Fig. 2
and results not shown). Neither CaCl2 nor Na2SO4 stimulated the enzyme.
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Biochemical (DeHertogh et al., 1964) and genetic studies (Charles & Aneja, 1999
) in B. japonicum and S. meliloti indicate that propionyl-CoA is metabolized via methylmalonyl-CoA to succinyl-CoA, which is then oxidized via the TCA cycle. We propose that R. etli utilizes the acyl-CoA carboxylase characterized here in this pathway, using propionyl-CoA generated via the degradation of certain amino acids or odd chain-length fatty acids as substrate. It is not known whether any of the methylmalonyl-CoA produced by the PCC reaction is used for the synthesis of secondary metabolites in rhizobia, although this is the major role of PCC in some prokaryotes (Kimura et al., 1998
; Rainwater & Kolattukudy, 1982
).
Evidence obtained from genome sequence analysis, protein Western blotting and the phenotypic characterization of the S. meliloti pccA mutant suggest that rhizobia possess an E. coli-type ACC which is presumably used for fatty acid synthesis. Because some bacterial ACC complexes are unstable in extracts prepared under normal conditions (Fall, 1976; Hector & Fall, 1976
; Guchait et al., 1974
), we compared ACC and PCC activities in strain CE3 extracts prepared by sonication in either CLB or the imidizole-ammonium sulfate buffer used to stabilize the ACC complex from Pseudomonas citronellolis (Hector & Fall, 1976
). No change in either ACC or PCC activity was observed in extracts prepared in the stabilizing buffer in comparison to those prepared in CLB, whereas if an additional ACC was stabilized under the former conditions, an increase in ACC activity would be expected. This result suggests that either the putative ACC complex of R. etli is markedly unstable and/or displays very low activity in vitro. Further genetic and biochemical studies, in progress, are necessary to define the physiological role of the R. etli acyl-CoA carboxylase characterized here.
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ACKNOWLEDGEMENTS |
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Received 22 September 2003;
revised 30 October 2003;
accepted 31 October 2003.
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