Zakharova Katerina, Law Jamison D, Gao Yuan, Kovvali Sravya, Wysocki Vicki H, Gopalan Venkat, Bell Charles E
Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, Ohio, USA.
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio, USA.
Protein Sci. 2025 Sep;34(9):e70260. doi: 10.1002/pro.70260.
The fra locus of Salmonella enterica encodes five genes for metabolism of fructose-asparagine, an Amadori product formed by condensation of asparagine with glucose. In the last step of this pathway, the FraB deglycase cleaves 6-phospho-fructose-aspartate into glucose-6-phosphate and aspartate. In homology models, FraB forms a homodimer with two equivalent active sites located at the dimer interface. E214 and H230, two invariant residues essential for catalysis, project into each active site cleft from opposing subunits of the dimer. Here, we have determined six crystal structures of FraB, three of a variant containing an N-terminal His tag and two mutations needed for crystallization (hereafter referred to as WT'), two with additional mutations to active site residues (E214A and P232A), and one of a variant with C-terminal residues 313-325 deleted. Surprisingly, in the WT' FraB structure, the two catalytic residues, E214 (general base) and H230 (general acid), are positioned ~22 Å apart. In the E214A and C-terminus-truncated FraB variants, however, a conformational change in the E214-residing helix brings E214 and H230* to ~7 Å (* indicates residue from the second protomer that creates the inter-subunit catalytic center). The loop bearing H230 also exhibits significant variation, ranging from being completely disordered to adopting open or closed states, with the nearby P232* residue being either cis or trans. The C-terminal residues 313-325 form a flexible "C-tail" that can be fully disordered, bind in the active site to block access of substrate, or angle across the active site to wrap across the other subunit of the dimer and potentially close over substrate. Collectively, these structures reveal that FraB is a conformational heterodimer with two chemically identical subunits that are constrained to adopt different structures as they come together for catalysis. This plasticity likely involves correlated opening and closure of the two active sites for their respective binding and release of substrates and ligands.
肠炎沙门氏菌的fra基因座编码五个参与果糖 - 天冬酰胺代谢的基因，果糖 - 天冬酰胺是天冬酰胺与葡萄糖缩合形成的一种阿马多里产物。在该途径的最后一步，FraB脱糖基酶将6 - 磷酸果糖 - 天冬氨酸裂解为6 - 磷酸葡萄糖和天冬氨酸。在同源模型中，FraB形成一个同型二聚体，两个等效的活性位点位于二聚体界面处。E214和H230是催化所必需的两个不变残基，从二聚体的相对亚基伸入每个活性位点裂隙中。在此，我们确定了FraB的六个晶体结构，其中三个是含有N端His标签和结晶所需的两个突变的变体（以下称为“WT'”），两个是活性位点残基有额外突变的结构（E214A和P'232A），还有一个是缺失C端残基313 - 325的变体。令人惊讶的是，在WT' FraB结构中，两个催化残基E214（通用碱）和H230（通用酸）相距约22 Å。然而，在E214A和C端截短的FraB变体中，位于E214的螺旋发生构象变化，使E214和H230*接近至约7 Å（表示来自第二个原体的残基，形成亚基间催化中心）。带有H230的环也表现出显著变化，范围从完全无序到呈现开放或封闭状态，附近的P232残基处于顺式或反式。C端残基313 - 325形成一个灵活的“C尾”，它可以完全无序，结合在活性位点以阻止底物进入，或者跨过活性位点以包裹二聚体的另一个亚基并可能封闭底物。总体而言，这些结构表明FraB是一种构象异二聚体，具有两个化学上相同的亚基，它们在聚集进行催化时被迫采用不同的结构。这种可塑性可能涉及两个活性位点各自结合和释放底物及配体时的相关开放和关闭。
