Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor
Catalytic mechanism of DcsB: Arginase framework used for hydrolyzing its inhibitor
复制标题
DOI：
10.1002/pro.4338
复制
发表时间：
2022-05
期刊：
影响因子：
8
通讯作者：
K. Oda;T. Sakaguchi;Y. Matoba
中科院分区：
文献类型：
--
作者：
K. Oda;T. Sakaguchi;Y. Matoba
DcsB是一种由d-环丝氨酸生物合成基因簇产生的酶，在序列和三维结构上与DcsB显示出中等程度的相似性。精氨酸酶是一种普遍存在的酶，水解L-精氨酸生成L-鸟氨酸和尿素，而DcsB水解Nω-羟基-L-精氨酸（L-NOHA）（一种精氨酸酶抑制剂）生成L-鸟氨酸和羟基脲。我们确定了与l-鸟氨酸和与2（S）-氨基-6-硼己酸的四面体衍生物缔合的DcsB的晶体结构，其硼原子与在活性中心桥接两个锰离子的氧原子形成共价键。DcsB的底物结合口袋比DcsB的底物结合口袋窄，表明DcsB不适合以抑制方式结合l-NOHA。过渡态样结构表明Asp 210和Glu 241具有捕获二锰簇附近带正电荷离子的作用。使用突变的DcsB的动力学分析表明，该酶在中性和碱性pH条件下采用不同的催化机制。DcsB中的Glu 241可能参与识别l-NOHA的羟基胍基，而Asp 210与Glu 241合作似乎有助于对质子化l-NOHA的反应性，这是中性pH条件下的优选物质。在质子化的l-NOHA进入DcsB的底物结合口袋后，水合氢离子可能被捕获在正离子结合位点。然后，该离子充当特定的酸催化剂，以促进l-NOHA的四面体中间体的崩溃。
DcsB, an enzyme produced from the d‐cycloserine biosynthetic gene cluster, displays moderate similarity to arginase in the sequence and three‐dimensional structure. Arginase is a ubiquitous enzyme hydrolyzing l‐arginine to generate l‐ornithine and urea, whereas DcsB hydrolyzes Nω‐hydroxy‐l‐arginine (l‐NOHA), an arginase inhibitor, to generate l‐ornithine and hydroxyurea. We determined the crystal structure of DcsB associated with l‐ornithine and that with the tetrahedral derivative of 2(S)‐amino‐6‐boronohexanoic acid, whose boron atom forms a covalent bond with an oxygen atom bridging two manganese ions at the active center. The substrate‐binding pocket of DcsB is narrower than that of arginase, suggesting that DcsB is unsuitable for the binding of l‐NOHA in an inhibitory manner. The transition state‐like structure demonstrated that Asp210 and Glu241 have a role to trap a positively charged ion near the dimanganese cluster. Kinetic analysis using the mutated DcsB showed that the enzyme employs different catalytic mechanisms under the neutral and alkaline pH conditions. Glu241 in DcsB is likely involved in the recognition of the hydroxyguanidino group of l‐NOHA, whereas Asp210, in cooperation with Glu241, seems to contribute to the reactivity toward the protonated l‐NOHA, which is a preferable species under the neutral pH conditions. After entering of the protonated l‐NOHA to the substrate‐binding pocket of DcsB, a hydronium ion may be trapped at the positive ion‐binding site. Then, the ion serves as a specific acid catalyst to facilitate the collapse of the tetrahedral intermediate of l‐NOHA.