Drago Victoria N, Devos Juliette M, Blakeley Matthew P, Forsyth V Trevor, Parks Jerry M, Kovalevsky Andrey, Mueser Timothy C
Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606, USA.
Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
Cell Rep Phys Sci. 2024 Feb 21;5(2). doi: 10.1016/j.xcrp.2024.101827. Epub 2024 Feb 12.
Pyridoxal 5'-phosphate (PLP), the biologically active form of vitamin B, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
磷酸吡哆醛（PLP）是维生素B的生物活性形式，是许多生物合成途径中必不可少的辅助因子。作为药物靶点和生物催化剂的PLP依赖性酶（如色氨酸合酶（TS））的出现，凸显了理解PLP依赖性催化和反应特异性的需求。中子衍射解析氢原子位置的能力使其成为了解PLP的静电环境和选择性质子化如何调节PLP依赖性活性的理想技术。在托莱多结晶盒的微重力结晶作用下，我们报道了处于内部醛亚胺形式的TS与PLP的2.1埃X射线/中子（XN）联合结构。在α和β活性位点（包括PLP辅因子）中直接确定了氢的位置。因此，联合XN结构为理解内部醛亚胺的选择性质子化以及了解整体催化机制所需的TS静电环境提供了见解。
