Gade Madhuri, Tan Li Lynn, Damry Adam M, Sandhu Mahakaran, Brock Joseph S, Delaney Andie, Villar-Briones Alejandro, Jackson Colin J, Laurino Paola
Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology Graduate University, 1919-1 Tancha, Onna 904-0495, Okinawa, Japan.
Research School of Chemistry, Australian National University, Canberra, 2601, Australia.
JACS Au. 2021 Nov 19;1(12):2349-2360. doi: 10.1021/jacsau.1c00464. eCollection 2021 Dec 27.
Protein conformational changes can facilitate the binding of noncognate substrates and underlying promiscuous activities. However, the contribution of substrate conformational dynamics to this process is comparatively poorly understood. Here, we analyze human (hMAT2A) and (eMAT) methionine adenosyltransferases that have identical active sites but different substrate specificity. In the promiscuous hMAT2A, noncognate substrates bind in a stable conformation to allow catalysis. In contrast, noncognate substrates sample stable productive binding modes less frequently in eMAT owing to altered mobility in the enzyme active site. Different cellular concentrations of substrates likely drove the evolutionary divergence of substrate specificity in these orthologues. The observation of catalytic promiscuity in hMAT2A led to the detection of a new human metabolite, methyl thioguanosine, that is produced at elevated levels in a cancer cell line. This work establishes that identical active sites can result in different substrate specificity owing to the effects of substrate and enzyme dynamics.
蛋白质构象变化可促进非同源底物的结合及潜在的混杂活性。然而，底物构象动力学对这一过程的贡献相对了解较少。在此，我们分析了人类（hMAT2A）和大肠杆菌（eMAT）的甲硫氨酸腺苷转移酶，它们具有相同的活性位点，但底物特异性不同。在具有混杂活性的hMAT2A中，非同源底物以稳定构象结合以进行催化。相比之下，由于酶活性位点的流动性改变，非同源底物在eMAT中较少采用稳定的有效结合模式。不同的细胞底物浓度可能推动了这些直系同源物底物特异性的进化分化。对hMAT2A中催化混杂性的观察导致检测到一种新的人类代谢物——甲基硫代鸟苷，其在癌细胞系中的水平升高。这项工作表明，由于底物和酶动力学的影响，相同的活性位点可导致不同的底物特异性。
