Preparative Synthesis of β-L-Malyl-coenzyme A Assisted by Malyl-coenzyme A Synthetase from Pseudomonas AM1
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β-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days. The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63 % homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences. Yields of β-L-[14C]malyl-CoA (1-10 μmol) were 70 % before and 65 % after purification in 0.1-0.5 μmol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column. For most biochemical work, the product was partially purified with an overall 45 % yield by chromatography on DEAE-Sephacel. The identity of the compound as β-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart. The enzymatic synthesis, especially of radioactively labeled β-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis.