The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis
The LarB carboxylase/hydrolase forms a transient cysteinyl-pyridine intermediate during nickel-pincer nucleotide cofactor biosynthesis
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DOI：
10.1073/pnas.2106202118
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发表时间：
2021-09-28
影响因子：
11.1
通讯作者：
Hausinger, Robert P.
中科院分区：
文献类型：
--
作者：
Rankin, Joel A.;Chatterjee, Shramana;Hausinger, Robert P.
具有镍钳核苷酸（NPN）辅因子的酶催化α-羟基酸底物的C2外消旋化或差向异构化反应。LarB通过进行双重反应：吡啶鎓环C5羧化和磷酸酐水解，从烟酸腺嘌呤二核苷酸（NaAD）开始合成NPN辅因子。在这里，我们表明，LarB使用二氧化碳，而不是碳酸氢盐，作为羧化的底物，并激活水的水解攻击AMP相关的磷酸盐的C5-羧化-NaAD。结构研究表明，LarB具有一个独特的折叠的N-末端结构域和一个C-末端结构域同源的氨基咪唑核糖核苷酸羧化酶/羧化酶（PurE）。与PurE一样，LarB是八聚体，具有位于亚基界面的四个活性位点。LarB与NAD*（NaAD的类似物）的复合物揭示了活性位点Cys 221和NAD* 的C4之间的共价加合物的形成，导致船形脱芳构化的吡啶环。这种中间体与NaAD的形成将增强C5的反应性以促进羧化。Glu 180很好地定位于提取C5质子，随着Cys 221被排出而恢复芳香性。分离的LarB及其与NAD* 和产物AMP的复合物的结构鉴定了对底物结合和催化潜在重要的另外的残基。结合这些发现，从结构指导的诱变研究的结果使我们提出的LarB的羧化和水解反应的酶机制是不同的PurE。
Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of alpha-hydroxyacid substrates. LarB initiates synthesis of the NPN cofactor from nicotinic acid adenine dinucleotide (NaAD) by performing dual reactions: pyridinium ring C5 carboxylation and phosphoanhydride hydrolysis. Here, we show that LarB uses carbon dioxide, not bicarbonate, as the substrate for carboxylation and activates water for hydrolytic attack on the AMP-associated phosphate of C5-carboxylated-NaAD. Structural investigations show that LarB has an N-terminal domain of unique fold and a C-terminal domain homologous to aminoimidazole ribonucleotide carboxylase/mutase (PurE). Like PurE, LarB is octameric with four active sites located at subunit interfaces. The complex of LarB with NAD*, an analog of NaAD, reveals the formation of a covalent adduct between the active site Cys221 and C4 of NAD*, resulting in a boat-shaped dearomatized pyridine ring. The formation of such an intermediate with NaAD would enhance the reactivity of C5 to facilitate carboxylation. Glu180 is well positioned to abstract the C5 proton, restoring aromaticity as Cys221 is expelled. The structure of as-isolated LarB and its complexes with NAD* and the product AMP identify additional residues potentially important for substrate binding and catalysis. In combination with these findings, the results from structure-guided mutagenesis studies lead us to propose enzymatic mechanisms for both the carboxylation and hydrolysis reactions of LarB that are distinct from that of PurE.