2-hydroxy acids are organic carboxylic acids ubiquitous in the living world and are important building blocks in organic synthesis. Recently, the lactate racemase (LarA) superfamily, a diverse superfamily of 2-hydroxy acid racemases and epimerases using the nickel-pincer nucleotide (NPN) cofactor, has been uncovered. In this study, we performed a taxonomic analysis of the LarA superfamily, showing the distribution of lactate racemase homologs (LarAHs) sequences across the three domains of life. Subsequently, we overexpressed and purified 9 LarAHs and investigated their biochemical properties and substrate specificities. We show that LarAHs from the lactate racemases group are more promiscuous than previously thought, with some members showing high specificity towards glycerate or 2-hydroxybutyrate. In other phylogenetic groups, we identified a new malate racemase and 2-hydroxyglutarate racemase, as well as a new 2-gluconate epimerase from an eukaryotic organism. We show that some LarAHs are able to isomerize up to 16 different substrates, mostly 2-hydroxy acids with hydrophobic side chains, thereby identifying 14 novel 2-hydroxy acid racemization and epimerization reactions catalyzed by LarAHs. These include the racemization of glycerate, 2-hydroxybutyrate, 2,4-dihydroxybutyrate, 2-hydroxyvalerate, 2-hydroxycaproate, 2,3-dihydroxyisovalérate, 2-hydroxy-3,3-dimethylbutyrate, 3-(4-hydroxyphenyl)lactate, 2-hydroxy-4-phenylbutyrate, and 2-hydroxy-4-oxo-4-phenylbutyrate. Additionally, we observed the C2-epimerization of all 2,3-dihydroxybutyrate stereoisomers (4-deoxy-DL-threonate and 4-deoxy-DL-erythronate) and the C2-epimerization of D-arabinarate epimers. Finally, through comparative analysis of Alphafold structural predictions, we identified key residues likely involved in substrate specificity and predicted the function of half of the LarAHs from the LarA superfamily. In conclusion, this study widely expands the scope of substrates isomerized by NPN-dependent enzymes.

中文翻译：

---

揭示了乳酸外消旋酶超家族中的 14 种新型 2-羟基酸外消旋化和差向异构化反应。


2-羟基酸是有机羧酸，在生物世界中无处不在，是有机合成的重要组成部分。最近，乳酸消旋酶 （LarA） 超家族被发现，这是一个使用镍钳形核苷酸 （NPN） 辅因子的 2-羟基酸消旋酶和差向异构酶的多样化超家族。在这项研究中，我们对 LarA 超家族进行了分类学分析，显示了乳酸消旋酶同源物 （LarAHs） 序列在生命的三个领域的分布。随后，我们过表达和纯化了 9 个 LarAHs，并研究了它们的生化性质和底物特异性。我们表明，来自乳酸外消旋酶组的 LarAHs 比以前认为的更混杂，一些成员对甘油酸或 2-羟基丁酸表现出高度特异性。在其他系统发育组中，我们从真核生物体中鉴定了一种新的苹果酸消旋酶和 2-羟基戊二酸消旋酶，以及一种新的 2-葡萄糖酸差向异构酶。我们表明，一些 LarAH 能够异构化多达 16 种不同的底物，主要是具有疏水侧链的 2-羟基酸，从而鉴定出 LarAH 催化的 14 种新型 2-羟基酸外消旋化和差向异构化反应。这些包括甘油酸酯、2-羟基丁酸酯、2,4-二羟基丁酸酯、2-羟基戊酸酯、2-羟基己酸酯、2,3-二羟基异戊酸酯、2-羟基-3,3-二甲基丁酸酯、3-（4-羟基苯基）乳酸酯、2-羟基-4-苯基丁酸酯和 2-羟基-4-氧代-4-苯基丁酸酯的外消旋化。此外，我们观察到所有 2,3-二羟基丁酸酯立体异构体 （4-脱氧-DL-苏糖酸酯和 4-脱氧-DL-赤藓酸酯） 的 C2-差向异构化和 D-阿拉伯酸差向异构体的 C2-差向异构化。 最后，通过对 Alphafold 结构预测的比较分析，我们确定了可能与底物特异性有关的关键残基，并预测了 LarA 超家族中一半 LarAHs 的功能。总之，本研究广泛扩展了 NPN 依赖性酶异构化的底物的范围。