title: Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.
abstract: Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using L-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93%, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 μmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg(2+) and was slightly stimulated by the monovalent cation K(+). It showed an improved thermostability, about 50% of the enzyme activity was retained even after preincubation at 50 °C for 2 h.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, People's Republic of China.
authors.forename: Gaofeng
authors.initials: G
authors.lastname: Yao
authors.forename: Xiulin
authors.initials: X
authors.lastname: Qin
authors.forename: Ju
authors.initials: J
authors.lastname: Chu
authors.forename: Xiaole
authors.initials: X
authors.lastname: Wu
authors.forename: Jiangchao
authors.initials: J
authors.lastname: Qian
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D032621:DNA Shuffling; D004926:Escherichia coli / Q000201:enzymology; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013306:Streptomycetaceae / Q000201:enzymology
chemical_list: D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D008715:Methionine; D008716:Methionine Adenosyltransferase