Lamer Tess, van Belkum Marco J, Chen Pu, Perov Ilia, Heard Bethan L, Wijewardane Anjalee, Lemieux M Joanne, Vederas John C
Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.
Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB T6G 2R3, Canada; Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada.
J Mol Biol. 2025 Aug 6;437(21):169375. doi: 10.1016/j.jmb.2025.169375.
D-amino acids play important roles in nature and are often produced from their L-stereoisomers by racemase or epimerase enzymes. One interesting class of amino acid racemases and epimerases are the cofactor-independent enzymes, which rely on a pair of active site cysteine residues for catalysis in an unusual chemical mechanism with seemingly mismatched acidity values. One classic example of these enzymes is diaminopimelic acid epimerase (DapF-CC), which produces D,L-diaminopimelic acid (DAP) as the penultimate step in lysine biosynthesis in most bacteria and photosynthetic organisms, and for Gram-negative bacterial peptidoglycan. In this work, we characterized for the first time enzymes of the cofactor-independent racemase and epimerase class that use paired catalytic serines (DapF-SS) instead of cysteines. DapF-SS enzymes catalyze reversible epimerization of DAP with similar kinetic parameters to that of DapF-CC enzymes. Sequence alignment and structural models suggest DapF-SS to have high homology to DapF-CC, and biochemical characterization provides evidence for a similar two-base mechanism. However, mutation of catalytic serine(s) to cysteine(s) nearly abolished activity, suggesting that these enzymes are not the result of simple mutations. A sequence similarity network identified thousands of other predicted DapF-SS enzymes from diverse bacterial phyla. Expression and isolation of several of these other enzymes found two with so far unidentified substrate(s), suggesting the Ser-Ser active site architecture may not be limited to just DAP epimerases. DapF-SS is active under oxidative conditions, while DapF-CC enzymes are inactivated by disulfide bond formation, providing a possible explanation as to why this second type of cofactor-independent epimerase evolved.
D-氨基酸在自然界中发挥着重要作用，通常由消旋酶或差向异构酶从其L-立体异构体产生。一类有趣的氨基酸消旋酶和差向异构酶是不依赖辅因子的酶，它们依靠一对活性位点半胱氨酸残基，通过一种具有看似不匹配酸度值的异常化学机制进行催化。这些酶的一个经典例子是二氨基庚二酸差向异构酶（DapF-CC），它在大多数细菌和光合生物的赖氨酸生物合成的倒数第二步中产生D，L-二氨基庚二酸（DAP），也是革兰氏阴性菌肽聚糖的组成成分。在这项工作中，我们首次对使用成对催化丝氨酸（DapF-SS）而非半胱氨酸的不依赖辅因子的消旋酶和差向异构酶类的酶进行了表征。DapF-SS酶催化DAP的可逆差向异构化，其动力学参数与DapF-CC酶相似。序列比对和结构模型表明DapF-SS与DapF-CC具有高度同源性，生化表征为类似的双碱基机制提供了证据。然而，将催化丝氨酸突变为半胱氨酸几乎消除了活性，这表明这些酶并非简单突变的结果。一个序列相似性网络从不同细菌门类中鉴定出数千种其他预测的DapF-SS酶。对其中几种其他酶的表达和分离发现，有两种酶的底物迄今尚未确定，这表明Ser-Ser活性位点结构可能不仅限于DAP差向异构酶。DapF-SS在氧化条件下具有活性，而DapF-CC酶会因二硫键形成而失活，这为第二种不依赖辅因子的差向异构酶为何进化提供了一种可能的解释。
