Enzymes possessing the nickel-pincer nucleotide (NPN) cofactor catalyze C2 racemization or epimerization reactions of α-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.

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 通过执行双重反应启动从烟酸腺嘌呤二核苷酸 (NaAD) 合成 NPN 辅因子：吡啶环 C5 羧化和磷酸酐水解。在这里，我们展示了 LarB 使用二氧化碳而不是碳酸氢盐作为羧化的底物，并激活水对 C5-羧化-NaAD 的 AMP 相关磷酸盐进行水解攻击。结构研究表明，LarB 具有一个独特折叠的 N 端结构域和一个与氨基咪唑核糖核苷酸羧化酶/变位酶 (PurE) 同源的 C 端结构域。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 ^{+的复合物NaAD 的类似物，揭示了在活性位点 Cys221 和 NAD +}的 C4 之间形成共价加合物，导致船形脱芳构化吡啶环。与 NaAD 形成这种中间体将增强 C5 的反应性以促进羧化。Glu180 可以很好地提取 C5 质子，在 Cys221 被排出时恢复芳香性。分离的 LarB 的结构及其与 NAD ⁺的复合物和产物 AMP 确定了对底物结合和催化可能重要的其他残基。结合这些发现，结构引导诱变研究的结果使我们提出了与 PurE 不同的 LarB 羧化和水解反应的酶促机制。