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. 2002 Nov;184(21):5999-6006.
doi: 10.1128/JB.184.21.5999-6006.2002.

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO(2) fixation

Sylvia Herter  1 , Andreas Busch, Georg Fuchs
Affiliations

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus, a bifunctional enzyme involved in autotrophic CO(2) fixation

Sylvia Herter et al. J Bacteriol. 2002 Nov.

Abstract

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.

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Figures

FIG. 1.
FIG. 1.
Proposed bicyclic pathway for autotrophic CO2 fixation in C. aurantiacus. (Left) 3-Hydroxypropionate cycle for CO2 fixation affording glyoxylate as first net CO2 fixation product. (Right) Proposed glyoxylate assimilation cycle. 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase; 3, propionyl-CoA synthase; 4, propionyl-CoA carboxylase; 5, epimerase; 6, methylmalonyl-CoA mutase; 7, citrate cycle enzymes (succinate dehydrogenase, fumarate hydratase); 8, succinyl-CoA:l-malate CoA transferase; 9, l-malyl-CoA lyase; 10, erythro-β-methylmalyl-CoA lyase; 11, postulated β-methylmalyl-CoA dehydratase; 12, postulated mesaconyl-CoA hydratase; 13, postulated citramalate activation by succinyl-CoA:l-citramalate CoA transferase; 14, postulated citramalyl-CoA lyase; 15, phosphoenolpyruvate carboxylase; 16, gluconeogenesis enzymes. erythro-β-methylmalyl-CoA, mesaconyl-CoA, and citramalate were identified as putative intermediates of the proposed glyoxylate assimilation cycle (18).
FIG. 2.
FIG. 2.
Organization of putative protein coding sequences (ORFs) on contig 965 (10.5 kb) of the C. aurantiacus strain J-10-fl genome. ytf1, putative CoA transferase (1.44 kb, 479 aa, 53 kDa); ytf2, putative CoA transferase (1.22 kb, 405 aa, 45 kDa); orf1, hypothetical protein of unknown function (0.64 kb, 231 aa, 23 kDa); ytf3, putative CoA transferase (1.23 kb, 409 aa, 45 kDa); mclA, putative malyl-CoA lyase gene (1.05 kb, 348 aa, 38 kDa); orf2, hypothetical protein of unknown function (1.06 kb, 352 aa, 38 kDa).
FIG. 3.
FIG. 3.
(A) Denaturing PAGE (12.5%) of purified recombinant l-malyl-CoA lyase of C. aurantiacus compared to purified β-methylmalyl-CoA lyase of C. aurantiacus (5 μg each). Lanes: 1, molecular mass marker (rabbit phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; egg ovalbumin, 45 kDa; lactate dehydrogenase, 34 kDa; carboanhydrase, 29 kDa; lysozyme, 14 kDa); 2, recombinant l-malyl-CoA lyase (size exclusion fraction); 3, purified β-methylmalyl-CoA lyase (Resource Q fraction) for comparison. The arrow indicates the corresponding molecular mass of around 38 kDa of both enzyme preparations. (B) Native PAGE (8%) of both enzyme preparations (5 to 10 μg of protein). Lanes: 1, molecular mass marker (bovine serum albumin monomer, 67 kDa; dimer, 134 kDa; trimer, 201 kDa; tetramer, 268 kDa); 2, recombinant l-malyl-CoA lyase (size exclusion fraction); 3, purified β-methylmalyl-CoA lyase (Resource Q fraction) for comparison. The arrow indicates a molecular mass of ≥210 kDa for both enzymes. The gels were stained with Coomassie brilliant blue R-250.
FIG. 4.
FIG. 4.
Denaturing PAGE (12.5%) of cell extract of autotrophically grown C. aurantiacus and β-methylmalyl-CoA lyase at various steps of purification (20 μg each). Lanes: 1, molecular mass marker (rabbit phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; egg ovalbumin, 45 kDa; lactate dehydrogenase, 34 kDa; carboanhydrase, 29 kDa); 2, cell extract; 3, cell extract after heat precipitation; 4, DEAE-Sepharose fraction; 5, phenyl-Sepharose fraction; 6, size exclusion fraction; 7, Resource Q fraction. The gel was stained with Coomassie brilliant blue R-250. The arrow indicates a 38-kDa protein band that corresponds to β-methylmalyl-CoA lyase.
FIG. 5.
FIG. 5.
Formation of acetyl-CoA and β-methylmalyl-CoA from l-malyl-CoA and propionyl-CoA by recombinant l-malyl-CoA lyase at 55°C. Results are shown for HPLC detection of CoA-thioesters after 0 min of incubation (A), after 20 min of incubation (B), or in a control experiment where l-malyl-CoA was omitted after 20 min of incubation (C). Retention times: 8 min (l-malyl-CoA), 10 min (β-methylmalyl-CoA; CoA), 16 min (acetyl-CoA), 24 min (propionyl-CoA). The reaction mixture (0.2 ml) contained 200 mM MOPS-K+ buffer (pH 7.7), 4 mM MgCl2, 2.5 mM l-malyl-CoA, 2.5 mM propionyl-CoA, and 24 μg of protein. Note that the l-malyl-CoA preparation contained 30% CoA as an impurity. Therefore, besides 2.5 mM l-malyl-CoA, 0.75 mM CoA was also initially present in the corresponding assay mixture. An unidentified product is indicated by the X.
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References

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