Evolution and Distribution of C7–Cyclitol Synthases in Prokaryotes and Eukaryotes

2-Epi-5-epi-valiolone synthase (EEVS), a C7-sugar phosphate cyclase (SPC) homologous to 3-dehydroquinate synthase (DHQS), was discovered during studies of the biosynthesis of the C7N-aminocyclitol family of natural products. EEVS was originally thought to be present only in certain actinomycetes, but analyses of genome sequences showed that it is broadly distributed in both prokaryotes and eukaryotes, including vertebrates. Another SPC, desmethyl-4-deoxygadusol synthase (DDGS), was later discovered as being involved in the biosynthesis of mycosporine-like amino acid sunscreen compounds. Current database annotations are quite unreliable, with many EEVSs reported as DHQS, and most DDGSs reported as EEVS, DHQS, or simply hypothetical proteins. Here, we identify sequence features useful for distinguishing these enzymes, report a crystal structure of a representative DDGS showing the high similarity of the EEVS and DDGS enzymes, identify notable active site differences, and demonstrate the importance of two of these active site residues for catalysis by point mutations. Further, we functionally characterized two representatives of a distinct clade equidistant from known EEVS and known DDGS groups and show them to be authentic EEVSs. Moreover, we document and discuss the distribution of genes that encode EEVS and DDGS in various prokaryotes and eukaryotes, including pathogenic bacteria, plant symbionts, nitrogen-fixing bacteria, myxobacteria, cyanobacteria, fungi, stramenopiles, and animals, suggesting their broad potential biological roles in nature.2-表-5-表-缬氨酮合酶（2-Epi-5-epi-valiolone synthase, EEVS）是一种C7糖磷酸环化酶（C7-sugar phosphate cyclase, SPC），与3-脱氢奎尼酸合酶（3-dehydroquinate synthase, DHQS）同源，该酶是在研究C7N-氨基环醇类天然产物的生物合成过程中被发现的。最初人们认为EEVS仅存在于某些放线菌中，但基因组序列分析显示，它广泛分布于原核生物和真核生物类群，包括脊椎动物。后续研究又发现了另一种SPC——去甲基-4-脱氧加杜尔合酶（desmethyl-4-deoxygadusol synthase, DDGS），其参与类菌胞素氨基酸类防晒化合物的生物合成。当前数据库注释存在较高的不可靠性：大量EEVS被注释为DHQS，而多数DDGS被注释为EEVS、DHQS，或仅标注为假想蛋白（hypothetical proteins）。本研究中，我们鉴定出可用于区分这两类酶的序列特征，解析了代表性DDGS的晶体结构，证实EEVS与DDGS酶之间具有高度相似性，同时发现了二者显著的活性位点差异，并通过点突变实验验证了其中两个活性位点残基对催化功能的重要性。进一步，我们对一个与已知EEVS和DDGS类群进化距离相当的独特进化分支的两个代表性酶进行了功能表征，证实它们属于真正的EEVS。此外，我们还梳理并讨论了编码EEVS和DDGS的基因在各类原核与真核生物中的分布情况，涵盖病原菌、植物共生菌、固氮菌、黏细菌、蓝细菌、真菌、不等鞭毛类以及动物类群，暗示这两类酶在自然界中具有广泛的潜在生物学功能。