Yang Janet G, Chen Hulda Yuchun, Guardado John H, Gardner Maile, Foronda Matthew S
Department of Chemistry, University of San Francisco, San Francisco, California, 94117.
Department of Chemistry, University of San Francisco, San Francisco, California, 94117.
J Biol Chem. 2025 Jul 17:110493. doi: 10.1016/j.jbc.2025.110493.
The Escherichia coli MetNI-Q importer, an ATP-binding cassette (ABC) transporter, mediates the uptake of both L- and D- enantiomers of methionine. Original in vivo uptake studies show a strong preference for L-Met over D-Met, but the molecular basis of this selectivity is unclear. In this work, we systematically examine substrate discrimination by the MetNI transporter and MetQ substrate binding protein using an array of biophysical and biochemical techniques. Based on the kinetic and thermodynamic parameters of individual intermediates in the transport cycle, we uncover multiple steps in the transport cycle that confer substrate specificity. As in many other ABC importer systems, selectivity is applied at the level of binding to the substrate binding protein: MetQ dictates a 1,000-fold preference for L-Met over D-Met. However, beyond this initial level of selectivity, MetQ displays distinct binding preferences for the MetNI transporter depending on the substrate. We propose that the differences in binding affinities reflect the more favored release of L-Met into the permeation pathway when compared to D-Met. In support of this model, under saturating conditions, MetNI transports L-Met across the lipid bilayer at a faster rate than D-Met. Interestingly, the ATPase activity of the MetNI-Q complex is not modulated by the presence of substrate. Our studies reveal that the MetNI-Q system incorporates two separate steps in tuning methionine uptake to substrate chirality and availability. This method of discrimination ensures the import of the most biologically preferred substrate while also allowing for adaptability to more limiting nutrient conditions.
大肠杆菌MetNI-Q转运体是一种ATP结合盒（ABC）转运蛋白，介导L-和D-对映体蛋氨酸的摄取。最初的体内摄取研究表明，该转运体对L-蛋氨酸的偏好远高于D-蛋氨酸，但这种选择性的分子基础尚不清楚。在这项研究中，我们使用一系列生物物理和生化技术，系统地研究了MetNI转运体和MetQ底物结合蛋白对底物的识别。基于转运循环中各个中间体的动力学和热力学参数，我们发现了转运循环中赋予底物特异性的多个步骤。与许多其他ABC进口转运体系统一样，选择性作用于与底物结合蛋白的结合水平：MetQ对L-蛋氨酸的偏好比对D-蛋氨酸高1000倍。然而，除了这种初始的选择性水平外，MetQ对MetNI转运体表现出取决于底物的独特结合偏好。我们认为，结合亲和力的差异反映了与D-蛋氨酸相比，L-蛋氨酸更倾向于释放到渗透途径中。支持这一模型的是，在饱和条件下，MetNI跨脂质双层转运L-蛋氨酸的速度比D-蛋氨酸快。有趣的是，MetNI-Q复合物的ATPase活性不受底物存在的调节。我们的研究表明，MetNI-Q系统在将蛋氨酸摄取调节至底物手性和可用性方面包含两个独立的步骤。这种识别方法确保了最符合生物学偏好的底物的导入，同时也允许适应更有限的营养条件。
