Department of Biochemistry and Centre de recherche en biologie structurale, McGill University, Montréal, QC H3G 0B1, Canada.
Laboratory of Organic Chemistry, ETH Zürich, CH-8093 Zürich, Switzerland.
Biochim Biophys Acta Gen Subj. 2022 Nov;1866(11):130217. doi: 10.1016/j.bbagen.2022.130217. Epub 2022 Jul 26.
Cyanophycinases are serine protease family enzymes which are required for the metabolism of cyanophycin, the natural polymer multi-L-arginyl-poly(L-aspartic acid). Cyanophycinases degrade cyanophycin to β-Asp-Arg dipeptides, which enables use of this important store of fixed nitrogen.
We used genetic code expansion to incorporate diaminopropionic acid into cyanophycinase in place of the active site serine, and determined a high-resolution structure of the covalent acyl-enzyme intermediate resulting from attack of cyanophycinase on a short cyanophycin segment.
The structure indicates that cyanophycin dipeptide residues P1 and P1' bind shallow pockets adjacent to the catalytic residues. We observe many cyanophycinase - P1 dipeptide interactions in the co-complex structure. Calorimetry measurements show that at least two cyanophycin dipeptides are needed for high affinity binding to cyanophycinase. We also characterized a putative cyanophycinase which we found to be structurally very similar but that shows no activity and could not be activated by mutation of its active site.
Despite its peptidic structure, cyanophycin is resistant to degradation by peptidases and other proteases. Our results help show how cyanophycinase can specifically bind and degrade this important polymer.
蓝藻菌精胺酶属于丝氨酸蛋白酶家族的酶，对于蓝藻菌精胺——天然多聚 L-精氨酸-多聚 L-天冬氨酸的代谢是必需的。蓝藻菌精胺酶将蓝藻菌精胺降解为β-Asp-Arg 二肽，从而使这种重要的固定氮源得以利用。
我们利用遗传密码扩展技术，将二氨基丙酸取代活性位点丝氨酸，整合到蓝藻菌精胺酶中，从而确定了蓝藻菌精胺酶攻击短蓝藻菌精胺片段后形成的共价酰基-酶中间产物的高分辨率结构。
该结构表明，蓝藻菌精胺二肽残基 P1 和 P1'结合在临近催化残基的浅口袋中。我们在共复合物结构中观察到许多蓝藻菌精胺酶-P1 二肽相互作用。量热法测量表明，至少需要两个蓝藻菌精胺二肽才能与蓝藻菌精胺酶高亲和力结合。我们还对一种假定的蓝藻菌精胺酶进行了表征，发现其结构非常相似，但没有活性，其活性位点的突变也不能使其激活。
尽管蓝藻菌精胺具有肽结构，但它能抵抗肽酶和其他蛋白酶的降解。我们的研究结果有助于解释蓝藻菌精胺酶如何特异性地结合和降解这种重要的聚合物。
