L -Malyl-Coenzyme A Lyase/ (cid:2) -Methylmalyl-Coenzyme A Lyase from Chloroﬂexus aurantiacus , a Bifunctional Enzyme Involved in Autotrophic CO 2 Fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO 2 ﬁxation pathway in the phototrophic Chlo- roﬂexus 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 (cid:1) -methylmalyl-CoA lyase to (cid:1) -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. (cid:1) -Methylmalyl-CoA lyase was puriﬁed from cell extracts of C. contained 200 mM MOPS-K (cid:1) buffer [pH 7.7], 4 mM MgCl 2 , 2 mM glyoxylate, 4 mM concentration of propionyl-CoA or acetyl-CoA, and protein. The reaction was started by the addition of protein. After 20 min of incubation, samples (25 (cid:5) l) were retrieved, diluted, and cooled down to room temperature by addition of 0.975 ml of 200 mM MOPS-K (cid:1) buffer (pH 7.4) containing 3.5 mM phenylhydrazinium chloride. After 15 min of incubation at room temperature, the formed glyoxylate phenylhydrazone was detected at 324 nm. In additional experiments, glyoxylate was replaced by pyruvate or oxaloacetate (2 mM concentration each), and acetyl-CoA or propionyl-CoA were replaced by succinyl-CoA. When the apparent K m values were to be determined, the concentration of the CoA-thioester or glyoxylate was varied between 0.05 and 20 mM; glyoxylate was either omitted (lyase reactions) or its concentration remained ﬁxed at 2 mM, and the samples (25 (cid:5) l) were developed after 5 min of incubation at 55°C (condensation reaction). Buffers used to determine the pH optimum were 200 mM MOPS-K (cid:1) , pH 6.0 to 8.9, at room temperature, which corresponded to pH 5.7 to 8.6 at 55°C; the (cid:4) pH of 0.3 was experimentally determined. The dependence of the reaction on divalent metal ions was investigated by addition of 1 mM EDTA to the reaction mixture in the absence of a divalent cation. The temperature optimum of the L -malyl-CoA lyase reaction was determined by varying the reaction temperature between 45 and 75°C. by TopLab (Martin- sried, Germany) using an Applied Biosystems Procise 492 sequencer (Weiter-stadt, Germany). The phenylthiohydantoin derivatives were identiﬁed with an online Applied Biosystems Analyzer 140 C. Optical absorption spectra of the puriﬁed (cid:2) -methylmalyl-CoA lyase from C. aurantiacus extract (0.44 mg of pro- tein ml (cid:6) 1 ) and of the puriﬁed recombinant L -malyl-CoA lyase from E. coli (2.4 mg of protein ml (cid:6) 1 ) were collected at room temperature using a Perkin Elmer Life Science Lambda 2S spectrometer and the same buffer as a blank.