The natural antivitamin 2′-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2′-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.

天然抗维生素2'-甲氧基-硫胺素（MTh）与抑制微生物生长有关。但是，其作用方式和酶选择性抑制机制仍然难以捉摸。有趣的是，MTh抑制某些硫胺素二磷酸（ThDP）酶，而在其他酶中具有辅酶活性。在这里我们报告了对大肠杆菌的强抑制作用MTh的转酮醇酶活性并阐明其作用方式及其结构基础。Mth二磷酸酯（MThDP）的独特2'-甲氧基与ThDP依赖性酶中辅因子激活所需的标准谷氨酸发生冲突。该谷氨酸被迫与相邻的谷氨酸形成稳定的抗催化低势垒氢键，从而破坏辅因子的活化。转酮酶和其他ThDP酶的分子动力学模拟将活性位点的灵活性和辅因子结合位点的拓扑结构确定为酶选择性抑制的关键决定因素。人的酶要么保留与MThDP的酶活性，要么优先结合真实的ThDP，而不是MThDP，而核心细菌代谢酶被抑制，证明具有治疗潜力。