The NAD(P)-dependent malate dehydrogenases (MDH) (EC 1.1.1.37) and NAD-dependent lactate dehydrogenases (LDH) (EC. 1.1.1.27) form a large superfamily that has been characterized in organisms belonging to the three Domains of Life. MDH catalyzes the reversible conversion of the oxaloacetate into malate, while LDH operates at the late stage of glycolysis by converting pyruvate into lactate. Phylogenetic studies proposed that the LDH/MDH superfamily encompasses five main groups of enzymes. Here, starting from 16,052 reference proteomes, we reinvestigated the relationships between MDH and LDH. We showed that the LDH/MDH superfamily encompasses three main families: MDH1, MDH2, and a large family encompassing MDH3, LDH, and L-2-hydroxyisocaproate dehydrogenases (HicDH) sequences. An in-depth analysis of the phylogeny of the MDH3/LDH/HicDH family and of the nature of three important amino acids, located within the catalytic site and involved in binding and substrate discrimination, revealed a large group of sequences displaying unexpected combinations of amino acids at these three critical positions. This group branched in-between canonical MDH3 and LDH sequences. The functional characterization of several enzymes from this intermediate group disclosed a mix of functional properties, indicating that the MDH3/LDH/HicDH family is much more diverse than previously thought, and blurred the frontier between MDH3 and LDH enzymes. Present-days enzymes of the intermediate group are a valuable material to study the evolutionary steps that led to functional diversity and emergence of allosteric regulation within the LDH/MDH superfamily.

NAD(P) 依赖性苹果酸脱氢酶 (MDH) (EC 1.1.1.37) 和 NAD 依赖性乳酸脱氢酶 (LDH) (EC. 1.1.1.27) 形成了一个大型超家族，其特征在于属于以下三个域的生物体。生活。MDH 催化草酰乙酸可逆地转化为苹果酸，而 LDH 在糖酵解后期通过将丙酮酸转化为乳酸来发挥作用。系统发育研究提出 LDH/MDH 超家族包含五个主要的酶组。在这里，我们从 16,052 个参考蛋白质组开始，重新研究了 MDH 和 LDH 之间的关系。我们发现 LDH/MDH 超家族包含三个主要家族：MDH1、MDH2 和一个包含 MDH3、LDH 和 L-2-羟基异己酸脱氢酶 (HicDH) 序列的大家族。对 MDH3/LDH/HicDH 家族的系统发育和位于催化位点内并参与结合和底物识别的三个重要氨基酸的性质的深入分析揭示了一大组序列显示出意想不到的氨基组合这三个关键位置的酸。该组在典型的 MDH3 和 LDH 序列之间分支。来自该中间组的几种酶的功能表征揭示了多种功能特性，表明 MDH3/LDH/HicDH 家族比以前认为的要多样化得多，并模糊了 MDH3 和 LDH 酶之间的界限。