Rosano C, Bisso A, Izzo G, Tonetti M, Sturla L, De Flora A, Bolognesi M
Department of Physics-INFM and Advanced Biotechnology Center-IST, University of Genova, Largo Rosanna Benzi 10, Genova, I-16132, Italy.
J Mol Biol. 2000 Oct 13;303(1):77-91. doi: 10.1006/jmbi.2000.4106.
GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.
GDP-4-酮-6-脱氧-D-甘露糖表异构酶/还原酶是一种双功能酶，负责GDP-L-岩藻糖生物合成的最后一步，而GDP-L-岩藻糖是岩藻糖基转移酶的底物。包括白细胞Lewis系统和病原菌细胞表面抗原在内的几种细胞表面抗原，其表达依赖于GDP-L-岩藻糖的可利用性。因此，该酶是依赖选择素介导的细胞间相互作用的病理状态下治疗的潜在靶点。先前的晶体学研究表明，GDP-4-酮-6-脱氧-D-甘露糖表异构酶/还原酶属于短链脱氢酶/还原酶蛋白同源家族。酶的活性位点区域位于N端NADPH结合结构域和C端结构域的界面处，后者用于结合底物。本文报道了GDP-4-酮-6-脱氧-D-甘露糖表异构酶/还原酶七种位点特异性突变体形式的设计、表达及功能特性。同时，基于同步加速器数据（R因子在12.6%至13.9%之间），对天然全酶和三个突变体（Ser107Ala、Tyr136Glu和Lys140Arg）的晶体结构进行了研究并在1.45-1.60 Å分辨率下进行了精修。精修后的蛋白质模型表明，除了活性位点残基Ser107、Tyr136和Lys140，其突变会损害整体酶活性并可能影响辅酶结合模式外，在预期底物（GDP-4-酮-6-脱氧-D-甘露糖）结合口袋周围能够进行质子交换的侧链，在表异构化和还原步骤中是选择性必需的。其中，Cys109和Hisl79可能在酶与表异构化催化中间体之间的质子交换中起主要作用。最后，分析了参与底物识别和酶稳定性的突变活性位点残基的额外作用。
