Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/86612
標題: Structure and mechanism of a nonhaem-iron SAM-dependentC-methyltransferase and its engineering to a hydratase and anO-methyltransferase
作者: Zou, Xiao-Wei
Liu, Yu-Chen
Hsu, Ning-Shian
Huang, Chuen-Jiuan
Lyu, Syue-Yi
Chan, Hsiu-Chien
Chang, Chin-Yuan
Yeh, Hsien-Wei
Lin, Kuan-Hung
Wu, Chang-Jer
Tsai, Ming-Daw
Li, Tsung-Lin
關鍵字: MppJ
methyltransferases
Crystallography, X-Ray
Iron
Methyltransferases
Models, Molecular
Protein Conformation
Protein Engineering
Streptomyces
摘要: In biological systems, methylation is most commonly performed by methyltransferases (MTs) using the electrophilic methyl source S-adenosyl-L-methionine (SAM) via the S(N)2 mechanism. (2S,3S)-β-Methylphenylalanine, a nonproteinogenic amino acid, is a building unit of the glycopeptide antibiotic mannopeptimycin. The gene product of mppJ from the mannopeptimycin-biosynthetic gene cluster is the MT that methylates the benzylic C atom of phenylpyruvate (Ppy) to give βMePpy. Although the benzylic C atom of Ppy is acidic, how its nucleophilicity is further enhanced to become an acceptor for C-methylation has not conclusively been determined. Here, a structural approach is used to address the mechanism of MppJ and to engineer it for new functions. The purified MppJ displays a turquoise colour, implying the presence of a metal ion. The crystal structures reveal MppJ to be the first ferric ion SAM-dependent MT. An additional four structures of binary and ternary complexes illustrate the molecular mechanism for the metal ion-dependent methyltransfer reaction. Overall, MppJ has a nonhaem iron centre that bind, orients and activates the α-ketoacid substrate and has developed a sandwiched bi-water device to avoid the formation of the unwanted reactive oxo-iron(IV) species during the C-methylation reaction. This discovery further prompted the conversion of the MT into a structurally/functionally unrelated new enzyme. Through stepwise mutagenesis and manipulation of coordination chemistry, MppJ was engineered to perform both Lewis acid-assisted hydration and/or O-methyltransfer reactions to give stereospecific new compounds. This process was validated by six crystal structures. The results reported in this study will facilitate the development and design of new biocatalysts for difficult-to-synthesize biochemicals.
URI: http://hdl.handle.net/11455/86612
ISSN: 1399-0047
1399-0047
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