Department of Chemistry, Michigan State University, East Lansing, Michigan, USA.
Department of Foodservice Management and Nutrition, College of Natural Sciences, Sangmyung University, Seoul, South Korea.
J Biol Chem. 2022 Jan;298(1):101395. doi: 10.1016/j.jbc.2021.101395. Epub 2021 Nov 8.
Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.
分支酶（BEs）在淀粉和糖原的生物合成中起着至关重要的作用，对这些聚合物的精细结构起着决定性作用。这些 BEs 的底物是长碳水化合物链，通过酶表面上的多个结合位点与这些酶相互作用。通过控制支链长度分布，BEs 可以调节淀粉和糖原部分的生理性质；然而，这种特异性的机制和结构决定因素仍然是神秘的。在这项研究中，我们在水稻 BE I（BEI）上鉴定出一个大的十二碳糖结合表面，它从活性位点的外部延伸到酶的活性位点。突变酶活性测定证实了该结合位点在酶催化中的重要性，由此我们得出结论，它可能是受体链结合位点。比较蓝藻和水稻 BE1 的结构，我们可以对供体结合位点进行建模。我们还鉴定了两个可能与供体链相互作用的环，其序列在倾向于转移较长链的植物 BE1 和仅转移更短链的 BEIIb 之间存在差异。当这些环的序列与 BEIIb 序列交换时，水稻 BE1 也变成了短链转移酶，证明了这些环在特异性中起着关键作用。总之，这些结果提供了 BEs 的结构、选择性和活性的更完整的图像。
