Tekel Andrej, Orságh Martin, Dračínský Martin, Pluskal Tomáš
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo náměstí 542/2, 160 00 Prague 6, Czech Republic.
Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Albertov 6, 120 00 Prague 2, Czech Republic.
ACS Omega. 2025 May 15;10(20):20509-20514. doi: 10.1021/acsomega.5c00855. eCollection 2025 May 27.
-adenosyl-l-methionine (SAM) is a crucial enzymatic cofactor that is conserved across all domains of life. Despite the pivotal role of this cofactor, its chirality at the sulfonium sulfur and the differing biological activities of its epimers, (,)-SAM and (,)-SAM, are often overlooked. Although enzymes predominantly utilize the (,)-SAM epimer, due to spontaneous epimerization at the sulfonium sulfur of SAM, the (,)-SAM epimer is present in all cells as well as in commercial SAM-containing products. Recently, an enzyme containing the DUF62 domain, identified as Salinispora tropica (StDUF62), has been shown to selectively hydrolyze (,)-SAM. It has been hypothesized that this function prevents the problematic accumulation of this epimer. Fluorinases, the only enzymes known to naturally incorporate fluorine into organic compounds, are homologous to enzymes of the DUF62 family. The discovery of unexpected diastereoselectivity of StDUF62 however raised an important question regarding the diastereoselectivity of the evolutionarily related bacterial fluorinase FlA1, an enzyme of significant importance. Given the relationship between these enzymes and their similar catalytic functions, it would be reasonable to hypothesize that FlA1 might also demonstrate activity toward the (,)-SAM diastereomer. Despite this homology, we report here the opposite diastereoselectivity of StDUF62 and Streptomyces sp. MA37 fluorinase (FlA1). The unusual lysine-free amino acid composition of StDUF62 suggests an evolutionary origin in haloadaptation; however, its SAM-hydrolyzing activity is greatly diminished at physiological concentrations of KCl or NaCl. We show that this inhibition is not caused solely by the competition with the chloride anion, as NaSO at equivalent ionic strength is also greatly diminishing StDUF62 activity, contrary to the fluorinating activity of FlA1. Both adenosine and increased ionic strength promoted StDUF62 trimer formation, whereas increased ionic strength alone led to inhibition. Considering the contrast between the wasteful hydrolysis of (,)-SAM and the energetically efficient mechanisms of eukaryotic (,)-SAM recycling, we suggest that (,)-SAM hydrolysis might not be the physiological function of StDUF62.
S-腺苷-L-甲硫氨酸（SAM）是一种关键的酶辅因子，在生命的所有领域中都保守存在。尽管这种辅因子具有关键作用，但其鎓硫处的手性以及其差向异构体（R）-SAM和（S）-SAM不同的生物活性常常被忽视。虽然酶主要利用（S）-SAM差向异构体，但由于SAM的鎓硫处会自发差向异构化，（R）-SAM差向异构体存在于所有细胞以及含SAM的商业产品中。最近，一种含有DUF62结构域的酶，被鉴定为热带盐单胞菌（StDUF62），已被证明能选择性地水解（R）-SAM。据推测，该功能可防止这种差向异构体出现问题性的积累。氟化酶是已知唯一能将氟自然掺入有机化合物的酶，与DUF62家族的酶同源。然而，StDUF62意外的非对映选择性的发现引发了一个关于进化相关的细菌氟化酶FlA1（一种非常重要的酶）的非对映选择性的重要问题。鉴于这些酶之间的关系及其相似的催化功能，合理推测FlA1可能也对（R）-SAM非对映体具有活性。尽管存在这种同源性，但我们在此报告了StDUF62和链霉菌属MA37氟化酶（FlA1）相反的非对映选择性。StDUF62异常的无赖氨酸氨基酸组成表明其在卤适应中有进化起源；然而，在生理浓度的KCl或NaCl下，其SAM水解活性会大大降低。我们表明这种抑制并非仅由与氯离子的竞争引起，因为在等效离子强度下，NaSO也会大大降低StDUF62的活性，这与FlA1的氟化活性相反。腺苷和增加的离子强度都促进了StDUF62三聚体的形成，而仅增加离子强度则导致抑制。考虑到（R）-SAM的浪费性水解与真核生物（S）-SAM循环的能量高效机制之间的对比，我们认为（R）-SAM水解可能不是StDUF62的生理功能。
