Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A

Michael F. Dunn, Gisela Araíza and Jaime Mora

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent Km and Vmax values of 0·064 mM and 2885 nmol min-1 (mg protein)-1, respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent Km values of 0·392 and 0·144 mM, respectively, and Vmax values of 423 and 268 nmol min-1 (mg protein)-1, respectively. K+, or Cs+ markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing {alpha} subunit and a 45 kDa biotin-free {beta} subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.


Abbreviations: ACC, acetyl-CoA carboxylase; PCC, propionyl-CoA carboxylase; PYC, pyruvate carboxylase


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Rhizobia belonging to the {alpha}-proteobacterial genera Rhizobium, Sinorhizobium, Bradyrhizobium, Mesorhizobium and Azorhizobium have the remarkable ability to fix atmospheric nitrogen in association with a compatible plant host. In addition to directly or indirectly regulating specific genes involved in a variety of metabolic processes (Encarnación et al., 2003; Heinz & Streit, 2003; Streit et al., 1996), the B-group vitamin biotin has profound effects on the physiology of rhizobia by acting as the prosthetic group in reactions catalysed by the biotin-dependent carboxylases (Encarnación et al., 1995; Lowe & Evans, 1962; Watson et al., 2001; West & Wilson, 1940). The function of biotin in these reactions is the activation and transfer of CO2 to an acceptor molecule such as pyruvate or an acyl-CoA, and organisms may contain one to several different biotin-dependent carboxylases. In Escherichia coli acetyl-CoA carboxylase (ACC; EC 6.4.1.2) is the sole biotin-dependent carboxylase, has absolute specificity for acetyl-CoA as an acceptor substrate (Alberts & Vagelos, 1968) and, because it catalyses the first committed step in fatty acid synthesis, is essential for viability. ACC is a heterotetramer composed of subunits encoded by the accA, accB, accC and accD genes (Cronan & Waldrop, 2002). In many cases, bacteria produce one or more acyl-CoA carboxylases able to use more than one acyl-CoA as a substrate. These enzymes are most properly designated as acyl-CoA carboxylases, but are sometimes called propionyl-CoA carboxylases (PCC; EC 6.4.1.3) or ACCs to reflect the substrate with which they have the most activity. The physiological roles of acyl-CoA carboxylases which have been delineated in bacteria include fatty acid synthesis (Rodríguez et al., 2001), degradation of branched-chain amino acids and odd chain-length fatty acids (Zubay, 1988), synthesis of polyketides derived from the carboxylation of acetyl-, propionyl- or butryryl-CoA (Hopwood & Sherman, 1990) and participation in the 3-hydroxypropionate cycle (Menendez et al., 1999). Most acyl-CoA carboxylase holo-enzymes are composed of equal numbers of {alpha} subunits (which contain biotin and are usually designated PccA) and {beta}-subunits (which do not contain biotin and are usually designated PccB) (Diacovich et al., 2002).

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 {alpha} and {beta} 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 {alpha} 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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains and growth media.
Bacterial strains used in this study are described in Table 1. Culture media and growth conditions were described previously (Dunn et al., 1996). Small-scale cell extracts for enzyme screenings were prepared by sonication as described previously (Dunn et al., 2001) using cells from PY (rich medium) cultures.


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Table 1. Strains used in this study

 
Acyl-CoA carboxylase assays.
PCC activity was measured using the 14CO2 incorporation assay (Dunn et al., 2002). With the purified acyl-CoA carboxylase preparation from R. etli, ACC and butyryl-CoA carboxylating activities were assayed identically with the exception of 3 mM acetyl-CoA or butryl-CoA, respectively, replacing propionyl-CoA. For the ACC screening assays of extracts from different rhizobia, acetyl-CoA was used at a final concentration of 1·5 mM. In all cases, reactions were initiated by the addition of the enzyme and incubated at 30 °C. At intervals, aliquots were withdrawn and acid-stable radioactivity was determined by liquid scintillation counting as described previously (Dunn et al., 2002).

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 30–55 % 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 200–700 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 0–400 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; {gamma}-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.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Acyl-CoA carboxylase activity in different rhizobia
Enzyme screenings for ACC and PCC activities were performed with extracts prepared from strains belonging to different members of the Rhizobiaceae (Table 2), many of which are either type strains or strains commonly used in rhizobial research (Table 1). Both PCC and ACC activities were present in all of the wild-type species examined, with PCC activity always being significantly higher (10–40-fold in wild-type strains) than that of ACC. The S. meliloti pccA mutant Rm11297 (Charles & Aneja, 1999) was essentially devoid of ACC and PCC activities (Table 2), providing the first biochemical confirmation of the function of the gene inactivated in this mutant. Consistent with these results we found that the mutant, unlike wild-type Rm1021, was incapable of growth on minimal medium containing propionate as sole carbon source (results not shown). This finding directly supports the contention that the S. meliloti pccA product is part of a functional acyl-CoA carboxylase which, acting in concert with the product of the methylmalonyl-CoA mutase (bhbA) encoded directly downstream, is part of a succinyl-CoA-producing anaplerotic pathway (Charles & Aneja, 1999; Miyamoto et al., 2003). It should be noted that this functionally indicative gene arrangement is absent in M. loti (Dunn et al., 2002a) and B. japonicum (Kaneko et al., 2002) where the putative pccA and pccB genes are neither encoded contiguously nor in the vicinity of bhbA. The fact that the S. meliloti pccA mutant is viable shows that the acyl-CoA carboxylase is not required for fatty acid synthesis.


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Table 2. Activities of PCC and ACC in some members of the Rhizobiaceae

 
Western blot analysis showed that all the rhizobia screened (Table 2) produced a biotin-containing protein, sometimes appearing as a doublet, which migrated in the 70–80 kDa range as well as a much smaller protein of approximately 14–20 kDa (results not shown). Contrary to expectations, the S. meliloti pccA mutant strain Rm11297 was not devoid of the 70–80 kDa protein, but produced significantly less of it than did wild-type strain Rm1021. It is plausible that the protein detected in this region of the blot is actually the product of the methylcrotonyl-CoA carboxylase {alpha} subunit-encoding gene localized on pSymB of S. meliloti (Dunn et al., 2002a). With the exception of B. japonicum and Azorhizobium caulinodans, all strains also produced an approximately 120 kDa band corresponding in size to the PYC subunit produced by R. etli and S. meliloti (Dunn et al., 1996, 2001).

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 {alpha} 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. 1a). 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 {alpha} subunit of the acyl-CoA carboxylase and the 45 kDa protein corresponds to the {beta} 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 {alpha}6{beta}6 enzymes. The experimentally determined molecular mass of the native R. etli acyl-CoA carboxylase fits fairly well with that calculated for an {alpha}6{beta}6 structure (714 000 Da).


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Table 3. Purification of the acyl-CoA carboxylase from R. etli strain 12-53

 


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Fig. 1. SDS-PAGE and Western blot analysis of the R. etli acyl-CoA carboxylase. (a) Coomassie-blue-stained SDS-PAGE gel of protein fractions obtained during the purification of the enzyme (see also Table 3). Lanes: 1, crude extract (40 µg total protein); 2, ammonium sulfate fraction (40 µg total protein); 3, DEAE Macroprep column eluate (20 µg total protein); 4, gel column eluate (10 µg total protein); 5, HIC column eluate (10 µg total protein); 6, BioScale Q2 column eluate (5 µg total protein). Positions of molecular mass standards are indicated on the left. The positions of the presumptive {alpha} and {beta} subunits of the enzyme are indicated. (b) Detection of a single biotinylated protein in the BioScale Q2 column eluate (0·7 µg total protein) following transfer to PVDF membrane and detection with streptavidin-HRP. The biotin-containing protein corresponds to the {alpha} subunit indicated in (a). (c) Determination of the native molecular mass of the purified acyl-CoA carboxylase on a BioSelect SEC400 gel permeation column. The protein profile (A280) and molecular mass of protein standards used for calibration is shown. A 1·1 µg sample of the enzyme was chromatographed and acyl-CoA carboxylase was detected by PCC assay.

 
Kinetic characteristics of the acyl-CoA carboxylase
Few prokaryotic acyl-CoA carboxylases have been studied with respect to both their kinetic properties and metal ion requirements. In reactions with propionyl-CoA as a substrate, the Km values of the R. etli enzyme for ATP and bicarbonate were 0·105 and 0·645 mM, respectively, and the S[50] for MgCl2 was 0·83 mM. The acyl-CoA substrate, MgCl2, ATP and bicarbonate were absolutely required for activity.

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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Table 4. Kinetic parameters of the R. etli acyl-CoA carboxylase with different acyl-CoA substrates

Units for Km and Vmax are mM and nmol min-1 (mg protein)-1, respectively. The specificity constant is calculated as Vmax/Km.

 
The activity of the partially purified enzyme with acetyl-CoA or propionyl-CoA as substrate was determined in the presence of potential metabolic effectors (up to 5 mM concentration). No substantial change in ACC or PCC activity by possible precursors of propionyl-CoA (L-valine, L-isoleucine, valerate or propionate) or acetyl-CoA (stearic acid, palmitic acid) was observed, nor did intermediates of the TCA cycle (succinate or citrate) affect the activities. ADP, a product of the reaction catalysed by biotin-dependent carboxylases, significantly inhibited the activity of both the ACC and PCC reactions (I[50] of 3 and 1 mM, respectively). Methylmalonyl-CoA, a product of the PCC reaction, gave I[50] values of 1·2 and >5 mM for the PCC and ACC reactions, respectively. Malonyl-CoA, a product of the ACC reaction, gave I[50] values of 4·5 and 1·8 mM for the PCC and ACC reactions, respectively. Thus the inhibition of the ACC and PCC activities of the acyl-CoA carboxylase was strongest with the corresponding acyl-CoA products.

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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Fig. 2. Activity of the PCC reaction of the acyl-CoA carboxylase as influenced by monovalent cations (chloride salts). Activities, determined as described in Methods, are the mean±SEM for duplicate assays.

 
The R. etli acyl-CoA carboxylase has a very high specificity constant for propionyl-CoA and essentially requires selected monovalent cations for activity. The acyl-CoA carboxylase from Myxococcus xanthus likewise displays a high specificity constant for propionyl-CoA, but is not activated by monovalent cations (Kimura et al., 1998). K+ only weakly activates the acyl-CoA carboxylase from Mycobacterium tuberculosis (Rainwater & Kolattukudy, 1982) and has no effect on the activity of the enzyme from Mycobacterium smegmatis, which is instead activated by (Haase et al., 1982). The ability of K+, and Cs+, but not Li+ or Na+, to activate the R. etli acyl-CoA carboxylase indicates that it is a member of the K+-requiring enzymes. The acyl-CoA carboxylase from the nematode Tubatrix aceti is another member of this group, in which K+ appears to increase the rate of biotin carboxylation rather than the rate of acyl-CoA carboxylation (Meyer & Meyer, 1978).

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.


   ACKNOWLEDGEMENTS
 
Partial financial support for this work was provided by grant 31711-N from CONACyT (Mexico) to M. F. D. We thank Drs T. C. Charles, E. Martínez-Romero and S. Brom for providing strains and Drs S. Encarnación and O. Geiger for valuable comments on the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 22 September 2003; revised 30 October 2003; accepted 31 October 2003.



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