School of Life Sciences, BK21 FOUR KNU Creative BioResearch Group, Kyungpook National University, Daegu 41566, Republic of Korea.
KNU Institute for Microorganisms, Kyungpook National University, Daegu 41566, Republic of Korea.
J Agric Food Chem. 2023 Oct 25;71(42):15692-15700. doi: 10.1021/acs.jafc.3c05180. Epub 2023 Oct 16.
S-Adenosylmethionine (SAM) acts as a methyl donor in living organisms, and S-adenosylmethionine synthetase (MetK) is an essential enzyme for cells, as it synthesizes SAM from methionine and adenosine triphosphate (ATP). This study determined the crystal structures of the apo form and adenosine/triphosphate complex form of MetK from (MetK). Results showed that MetK has an allosteric inhibitor binding site for the SAM product in the vicinity of the active site and is inhibited by SAM both competitively and noncompetitively. Through structure-guided protein engineering, the MetK variant was developed that exhibited an almost complete release of inhibition by SAM with rather enhanced enzyme activity. The crystal structure of the MetK variant revealed that the formation of a new hydrogen bond between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and also improved the protein thermal stability by strengthening the tetramerization of the enzyme.
S-腺苷甲硫氨酸（SAM）在生物体内作为甲基供体发挥作用，而 S-腺苷甲硫氨酸合成酶（MetK）是细胞内的必需酶，因为它可以将甲硫氨酸和三磷酸腺苷（ATP）合成 SAM。本研究测定了 （MetK）的 apo 形式和腺苷/三磷酸复合物形式的 MetK 晶体结构。结果表明，MetK 在活性位点附近具有 SAM 产物的别构抑制剂结合位点，并且受到 SAM 的竞争性和非竞争性抑制。通过结构导向的蛋白质工程，开发出的 MetK 变体几乎完全释放了 SAM 的抑制作用，同时酶活性得到了显著提高。MetK 变体的晶体结构表明，E68A 突变形成的 Tyr66 和 Glu102 之间新的氢键破坏了别构 SAM 结合位点，并且通过增强酶的四聚化提高了蛋白质的热稳定性。
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