It is well known that, in organisms, one of the functions of enzymes is to avoid the accumulation of bioorganic compounds that can inhibit biological processes indispensable for cell function. Recently, it has been found that organisms such as yeast, fruit flies, nematodes and plants use a methyltransferase enzymes to convert isomer R-Adenosyl-L-methionine to its bioactive form S-adenosyl-L-methionine (AdoMet). This metabolite is the second most widely used substrate in all phyla of life and is involved in reactions essential for cellular biochemistry. The diversity of functionalities present in the metabolite explain its consumption in processes as the recycling of methionine, peptide and tRNA modification, lipid metabolism, metalloprotein cluster formation, among others. Particularly, yeast employs two homocysteine methyltransferase Sam4 and Mht1 (EC 2.1.1.10) to deplete R-AdoMet accumulation. Even though no disease has been direclty associate to the presence of R-AdoMet, its accumulation can be toxic to celullar processes, as its presence occupy valuable intracelullar space. In humans, the pathways that carry out the conversion of R-AdoMet to S-Adomet still remain elusive. Therefore, the focus of the study is to express and purify recombinant Sam4 in Escherichia coli by immobilized metal affinity chromatography. These procedure would help to obtain large amounts of the recombinant protein in order to study its enzymatic capacity by using AdoMet derivatives, which will potentially the study of the catalytic mechanism. First, the method was applied to the recombinant human thiopurine methyltransferase proteins (TPMT, EC 2.1.1.67), and the method results effective since a high yield and reproducible purification was observed. TPMT catalysis shows that a methyl transfer to aromatic thiols occurs in a time and enzyme concentration dependent manner. Sam4 expression was performed successfully, but the purification was partially effective. Consequently, the study of the enzymatic capacity of Sam4 shows a minimum activity at the condition employed. The results imply that the purification method should be refined. The affinity purification can be optimized in Sam4 studies to fully understand and decrypt the enzyme mechanism and functions in vivo, to detect substrates that are catalyzed by it, and to elucidate the physiological response of R-AdoMet bioacumulation.

Expression and purification of homocysteine methyltransferase Sam4 recombinant in Escherichia coli

249th National Meeting (2015)

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