Preparative synthesis of beta-L-malyl-coenzyme A assisted by malyl-coenzyme A synthetase from Pseudomonas AM1.
beta-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 beta-L-[14C]malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol 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 beta-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 beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis.
Willibald
B
B
Institut für Biophysik und physikalische Biochemie der Universität, Universität Regensburg, Germany.
Boves
H
H
Holler
E
E
D000214:Acyl Coenzyme A / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D003066:Coenzyme A Ligases / Q000302:isolation & purification*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification; D011549:Pseudomonas / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; C042519:malyl-coenzyme A; D007652:Oxo-Acid-Lyases; C021536:malyl-CoA lyase; D003066:Coenzyme A Ligases; C021537:malate - CoA ligase