Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.
Cytiva, Marlborough, Massachusetts 01752, United States.
Biochemistry. 2020 Jul 28;59(29):2751-2759. doi: 10.1021/acs.biochem.0c00432. Epub 2020 Jul 12.
Farnesyl diphosphate synthase (FPPS) is an isoprenoid chain elongation enzyme that catalyzes the sequential condensation of dimethylallyl diphosphate (C) with isopentenyl diphosphate (IPP; C) and the resulting geranyl diphosphate (GPP; C) with another molecule of IPP, eventually producing farnesyl diphosphate (FPP; C), which is a precursor for the biosynthesis of a vast majority of isoprenoids. Previous studies of FPPS have highlighted the importance of the structure around the hydrophobic chain elongation path in determining product specificity. To investigate what structural features define the final chain length of the product in FPPS from , we designed and expressed six mutants of FPPS by replacing small amino acids around the binding pocket with bulky residues. Using enzymatic assays, binding kinetics, and crystallographic studies, we analyzed the effects of these mutations on the activity and product specificity of FPPS. Our results revealed that replacement of Thr-164 with tryptophan and phenylalanine completely abolished the activity of FPPS. Intriguingly, the T164Y substitution displayed dual product specificity and produced a mixture GPP and FPP as final products, with an activity for FPP synthesis that was lower than that of the wild-type enzyme. These data indicate that Thr-164 is a potential regulator of product specificity.
法呢基二磷酸合酶（FPPS）是一种异戊烯链延伸酶，可催化二甲基烯丙基二磷酸（C）与异戊烯二磷酸（IPP；C）的顺序缩合，以及随后的香叶基二磷酸（GPP；C）与另一个 IPP 分子的缩合，最终产生法呢基二磷酸（FPP；C），这是大多数异戊烯类生物合成的前体。之前对 FPPS 的研究强调了围绕疏水性链延伸路径的结构在确定产物特异性方面的重要性。为了研究什么结构特征定义了来自 的 FPPS 产物的最终链长，我们通过用大体积残基替换结合口袋周围的小氨基酸设计并表达了 FPPS 的六个突变体。通过酶促测定、结合动力学和晶体学研究，我们分析了这些突变对 FPPS 活性和产物特异性的影响。我们的结果表明，用色氨酸和苯丙氨酸替换 Thr-164 完全消除了 FPPS 的活性。有趣的是，T164Y 取代显示出双重产物特异性，并产生 GPP 和 FPP 的混合物作为最终产物，其 FPP 合成活性低于野生型酶。这些数据表明 Thr-164 是产物特异性的潜在调节剂。
