硫胺素
立体化学
裂解酶
化学
活动站点
序列母题
辅因子
酶
位阻效应
脱羧
结构母题
生物化学
催化作用
DNA
作者
Lucrezia Lanza,Fabian Rabe von Pappenheim,Daniela Bjarnesen,Camilla Leogrande,Alexandra Paul,Leonhard Krug,Kai Tittmann,Michael Müller
标识
DOI:10.1002/anie.202404045
摘要
The thiamine diphosphate (ThDP)‐binding motif, characterized by the canonical GDG(X)24‐27N sequence, is highly conserved among ThDP‐dependent enzymes. We investigated a ThDP‐dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. We found that JanthE has a high substrate promiscuity accepting long aliphatic α‐keto acids as donors. Sterically hindered aromatic aldehydes or non‐activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in the cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. We further determined the long‐sought genuine tetrahedral intermediates formed with pyruvate and 2‐oxo‐butyrate in the pre‐decarboxylation states and unravel atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP‐binding motif and unlock a family of lyases able to perform valuable carboligation reactions.
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