Yuan Yue, Bleile Dustin W, Wen Xin, Sanders David A R, Itoh Kenji, Liu Hung-wen, Pinto B Mario
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6.
J Am Chem Soc. 2008 Mar 12;130(10):3157-68. doi: 10.1021/ja7104152. Epub 2008 Feb 16.
UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDP-Galp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a manner similar to that of UDP-Galp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the "open" mobile loop in UGM "closes" upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf and shed light on the mechanism of action of UGM.
尿苷二磷酸 -α-D-吡喃半乳糖变位酶（UGM）是参与Galf生物合成的关键酶。尿苷二磷酸 -α-D-吡喃半乳糖（UDP-Galp）和尿苷二磷酸 -α-L-吡喃半乳糖（UDP-Galf）是UGM的两种天然底物。一种结合使用STD-NMR光谱、分子建模和CORCEMA-ST计算的方法被应用于研究UDP-Galf及其C3-氟化类似物与肺炎克雷伯菌UGM的结合。UDP-Galf和UDP-[3-F]Galf以类似于UDP-Galp的方式与UGM结合。通过NMR光谱观察到，还原态（活性）的UGM催化UDP-Galf和UDP-[3-F]Galf向其吡喃半乳糖对应物的相互转化，且催化效率不同。通过竞争STD-NMR实验还比较了UDP-Galf和UDP-[3-F]Galf与UDP-Galp和UDP的结合亲和力。当UGM处于氧化态（无活性）时，UDP-Galf、UDP-Galp和UDP-[3-F]Galf的结合亲和力大小相似且低于UDP。然而，当UGM处于还原态时，与UDP相比，UDP-Galp具有更高的结合亲和力。分子动力学（MD）模拟表明，UGM中的“开放”移动环在底物结合时会“关闭”。结合MD模拟和STD-NMR实验创建了以UDP-Galf和UDP-[3-F]Galf作为结合配体的UGM模型。将CORCEMA-ST（饱和转移的完全弛豫和构象交换矩阵分析）计算的饱和转移效应值与实验STD效应进行比较，从而区分结合配体的两个主要构象家族。综上所述，这些结果用于解释UDP-Galf和UDP-[3-F]Galf不同的催化周转速率，并阐明UGM的作用机制。
