Synthesis of alpha, beta-deuterated 15N amino acids using a coupled glutamate dehydrogenase-branched-chain amino acid aminotransferase system.
Gram amounts of various 15N-enriched L-amino acids have been synthesized using a coupled enzymatic system. Catalytic amounts of 15N-labeled L-glutamate are generated using (15NH4)2SO4, alpha-ketoglutarate, and glutamate dehydrogenase. The labeled glutamate in turn serves as an amine donor to an appropriate alpha-keto acid using the Escherichia coli branched-chain amino acid aminotransferase, the subcloning and overexpression of which is described. In order to drive the reaction to completion the redox cofactor NADPH is regenerated using a glucose dehydrogenase system. Since the amino-transferase catalyzes exchange of the alpha-hydrogen, carrying out this reaction in 2H2O gives rise to 2-2H,2-15N-labeled amino acids. Deuteration can be readily extended to the beta position as well by preexchanging the alpha-keto acids in basic 2H2O. The isotopically labeled amino acids are recovered in yields of 70-80%.
Chanatry
J A
JA
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208.
Schafer
P H
PH
Kim
M S
MS
LeMaster
D M
DM
D000596:Amino Acids / Q000138:chemical synthesis*; D017123:Bacteriophage T7 / Q000201:enzymology; D002021:Buffers; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003903:Deuterium; D005822:Genetic Vectors / Q000235:genetics; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D009587:Nitrogen Isotopes; D000637:Transaminases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D002021:Buffers; D009587:Nitrogen Isotopes; D003903:Deuterium; D005969:Glutamate Dehydrogenase; D000637:Transaminases; C019798:branched-chain-amino-acid transaminase; D012321:DNA-Directed RNA Polymerases