Enzymatic synthesis of S-adenosyl-L-methionine on the preparative scale.
The problems inherent in the enzymatic and chemical synthesis of S-adenosyl-L-methionine (SAM) led us to develop an efficient, simple method for the synthesis of large amounts of labeled SAM. Previously, we reported that the problem of product inhibition of E. coli SAM synthetase encoded by the metK gene was successfully overcome in the presence of sodium p-toluenesulfonate (pTsONa). This research has now been expanded to demonstrate that product inhibition of this enzyme can also be overcome by adding a high concentration of beta-mercaptoethanol (beta ME), acetonitrile, or urea. In addition a recombinant strain of E. coli has been constructed that expresses the yeast SAM synthetase encoded by the sam2 gene. The yeast enzyme does not have the problem of product inhibition seen with the E. coli enzyme. Complete conversion of 10 mM methionine to SAM was achieved in incubations with either the recombinant yeast enzyme and 1 molar potassium ion or the E. coli enzyme in the presence of additives such as beta ME, acetonitrile, urea, or pTsONa. The recombinant yeast SAM synthetase was used to generate SAM in situ for use in the multi-enzymatic synthesis of precorrin 2.
Park
J
J
Center for Biological NMR, Department of Chemistry, Texas A&M University, College Station 77843-3255, USA.
Tai
J
J
Roessner
C A
CA
Scott
A I
AI
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008716:Methionine Adenosyltransferase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000096:biosynthesis*; D012492:Salts; D012997:Solvents; D014578:Uroporphyrins / Q000096:biosynthesis; D015003:Yeasts / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D012492:Salts; D012997:Solvents; D014578:Uroporphyrins; D012436:S-Adenosylmethionine; C063209:15,23-dihydrosirohydrochlorin; D008716:Methionine Adenosyltransferase