PDF(Pubmed)
Abstract：
Enzymatic transglycosylation of the fleximer base 4-(4-aminopyridine-3-yl)-1H-pyrazole using recombinant E. coli purine nucleoside phosphorylase (PNP) resulted in the formation of \"non-typical\" minor products of the reaction. In addition to \"typical\" N1-pyrazole nucleosides, a 4-imino-pyridinium riboside and a N1-pyridinium-N1-pyrazole bis-ribose derivative were formed. N1-Pyrazole 2\'-deoxyribonucleosides and a N1-pyridinium-N1-pyrazole bis-2\'-deoxyriboside were formed. But 4-imino-pyridinium deoxyriboside was not formed in the reaction mixture. The role of thermodynamic parameters of key intermediates in the formation of reaction products was elucidated. To determine the mechanism of binding and activation of heterocyclic substrates in the E. coli PNP active site, molecular modeling of the fleximer base and reaction products in the enzyme active site was carried out. As for N1-pyridinium riboside, there are two possible locations for it in the PNP active site. The presence of a relatively large space in the area of amino acid residues Phe159, Val178, and Asp204 allows the ribose residue to fit into that space, and the heterocyclic base can occupy a position that is suitable for subsequent glycosylation. Perhaps it is this \"upside down\" arrangement that promotes secondary glycosylation and the formation of minor bis-riboside products.
摘要：
使用重组大肠杆菌嘌呤核苷磷酸化酶（PNP）对fleximer碱基4-（4-氨基吡啶-3-基）-1H-吡唑进行酶促糖基化，导致形成“非典型”次要反应产物。除了“典型的”N1-吡唑核苷，形成4-亚氨基-吡啶鎓核苷和N1-吡啶鎓-N1-吡唑双核糖衍生物。形成N1-吡唑2'-脱氧核糖核苷和N1-吡啶-N1-吡唑双-2'-脱氧核苷。但是在反应混合物中没有形成4-亚氨基-吡啶鎓脱氧核苷。阐明了关键中间体的热力学参数在反应产物形成中的作用。为了确定大肠杆菌PNP活性位点中杂环底物的结合和活化机制，对酶活性位点的fleximer碱基和反应产物进行了分子建模。至于N1-吡啶核苷，在PNP活性位点有两个可能的位置。在氨基酸残基Phe159、Val178和Asp204的区域中相对较大的空间的存在允许核糖残基适合该空间,并且杂环碱基可以占据适于随后糖基化的位置。也许正是这种“颠倒”的排列促进了次级糖基化和少量双核苷产物的形成。
