Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum.
The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.
Freudenberg
W
W
Institut für Mikrobiologie der Universität, Göttingen, Federal Republic of Germany.
Andreesen
J R
JR
D000594:Amino Acid Oxidoreductases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D050960:Aminomethyltransferase; D003429:Cross Reactions; D005051:Eubacterium / Q000201:enzymology*; D050841:Glycine Dehydrogenase (Decarboxylating); D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D013057:Spectrum Analysis; D008063:Thioctic Acid / Q000032:analysis; D014166:Transferases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D009097:Multienzyme Complexes; D009243:NAD; D008063:Thioctic Acid; D000594:Amino Acid Oxidoreductases; D050841:Glycine Dehydrogenase (Decarboxylating); D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase