Threonine formation via the coupled activity of 2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase.
The enzymes L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A (CoA) lyase are known to catalyze the net conversion of L-threonine plus NAD+ plus CoA to NADH plus glycine plus acetyl-CoA. When homogeneous preparations of these two enzymes from Escherichia coli were incubated together for 40 min at 25 degrees C with glycine, acetyl-CoA, and NADH, a 36% decrease in the level of glycine (with concomitant NADH oxidation) was matched by formation of an equivalent amount of threonine, indicating that this coupled sequence of enzyme-catalyzed reactions is reversible in vitro. Several experimental factors that affect the efficiency of this conversion in vitro were examined. A constructed strain of E. coli, MD901 (glyA thrB/C tdh), was unable to grow unless both glycine and threonine were added to defined rich medium. Introduction of the plasmid pDR121 (tdh+kbl+) into this strain enabled the cells to grow in the presence of either added glycine or threonine, indicating that interconversion of these two amino acids occurred. Threonine that was isolated from the total pool of cellular protein of MD901/pDR121 had the same specific radioactivity as the [14C]glycine added to the medium, establishing that threonine was formed exclusively from glycine in this strain. Comparative growth rate studies with several strains of E. coli containing plasmid pDR121, together with the finding that kcat values of pure E. coli 2-amino-3-ketobutyrate CoA lyase favor the cleavage of 2-amino-3-ketobutyrate over its formation by a factor of 50, indicate that the biosynthesis of threonine is less efficient than glycine formation via the coupled threonine dehydrogenase-2-amino-3-ketobutyrate lyase reactions.
Marcus
J P
JP
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Dekker
E E
EE
D000096:Acetone / Q000031:analogs & derivatives / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D000123:Acetyltransferases / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D003065:Coenzyme A / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D013912:Threonine / Q000096:biosynthesis*
D000096:Acetone; D013912:Threonine; D000105:Acetyl Coenzyme A; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase; D000123:Acetyltransferases; C020679:glycine acetyltransferase; D003065:Coenzyme A; D005998:Glycine; C006495:aminoacetone