Biosynthesis of isotopically enriched l-serine

Abstract

Isotopically enriched amino acids have a wide range of uses in biochemistry, e.g. I3C enrichment can be utilized for 13C nuclear magnetic resonance (n.m.r.) studies of enzymes (Malthouse, 1986). Isotopically labelled serine could also be used as precursor for the synthesis of other amino acids. Most chemical o r enzymic procedures have focused on preparing deuterium-or tritium-enriched serine and either produce low yields (less than 20%) or poor stereospecificity (Slieker & Benkovic, 1982). Consequently, isotopically enriched serine is expensive, e.g. I3C-enriched L-serine is at *To whom correspondence should he addressed. A 3

Enzymatic synthesis of isotopically labelled serine and tryptophan for application in peptide synthesis

“…The production of serine under these conditions with the E. coli enzyme approximated to a first order process with a half life of 2 h as was observed with the beef liver enzyme [9,10]. On adding serine hydroxymethyltransferase to the incubation mixture (see Experimental Part) there was a progressive decrease in intensity of the signals due to [1,[2][3][4][5][6][7][8][9][10][11][12][13] Tryptophan synthase catalyses the formation of tryptophan from indole and serine [17]. We have used tryptophan synthase from Salmonella typhimurium to effect the formation of [1,2-13 C 2 , 15 N]tryptophan from [1,2-13 C 2 , 15 N]serine and indole (see Experimental Part).…”

“…The experimental conditions are essentially the same, except that the concentrations were increased: pyridoxal phosphate from 0.06 to 0.1 mM, formaldehyde from 30 to 50 mM, active enzyme from 1.2 to 8.0 mM (this corresponds to increasing the catalytic activity of the enzyme from 0.14 to 0.69 mmol/ml/min, using L-allothreonine as a substrate [16]); the concentration of 2-mercaptoethanol was decreased from 10 to 5 mM. The production of serine under these conditions with the E. coli enzyme approximated to a first order process with a half life of 2 h as was observed with the beef liver enzyme [9,10]. On adding serine hydroxymethyltransferase to the incubation mixture (see Experimental Part) there was a progressive decrease in intensity of the signals due to [1,[2][3][4][5][6][7][8][9][10][11][12][13] Tryptophan synthase catalyses the formation of tryptophan from indole and serine [17].…”

“…In this work we have applied an enzymatic procedure involving serine hydroxymethyltransferase (EC 2.1.2.1.) developed in the Dublin laboratory, which was first applied several years ago to make [2-13 C]serine on a small scale but only briefly reported then [9,10]. Full experimental details of this procedure will now be given.…”

“…Serine hydroxymethyltransferase catalyses the formation of serine from glycine using 5,10-methylenetetrahydrofolate as a C 1 donor [15]. Serine hydroxymethyltransferase from beef liver has been used to effect the synthesis of [2-13 C]serine from [2-13 C]glycine [9,10]. In the present work serine hydroxymethyltransferase from E. coli was used to catalyse the synthesis of [1,2-13 C 2 , 15 N]serine from [1,2-13 C 2 , 15 N]glycine.…”

“…On adding tryptophan synthase to the reactants, there was a progressive decrease in intensity of the signals due to [1,2-13 C 2 , 15 N]serine and a concomitant increase of those due to [1,2-13 C 2 , 15 N]tryptophan (56.0 (dd, J C,C 53.8, J C,N 6.1, a-13 C), 175.4 (d, J C,C 53.8, 13 COOH)). After 48 h at 37 C, $ 95% of the [1,2-13 C 2 , 15 N]serine had been converted to [1,[2][3][4][5][6][7][8][9][10][11][12][13] …”