Davis Tony D, Kunakom Sylvia, Burkart Michael D, Eustaquio Alessandra S
University of California San Diego, San Diego, CA, United States.
University of Illinois at Chicago, College of Pharmacy, and Center for Biomolecular Sciences, Chicago, IL, United States.
Methods Enzymol. 2018;604:367-388. doi: 10.1016/bs.mie.2018.02.012. Epub 2018 Apr 2.
S-adenosyl-l-methionine (SAM) is universal in biology, serving as the second most common cofactor in a variety of enzymatic reactions. One of the main roles of SAM is the methylation of nucleic acids, proteins, and metabolites. Methylation often imparts regulatory control to DNA and proteins, and leads to an increase in the activity of specialized metabolites such as those developed as pharmaceuticals. There has been increased interest in using SAM analogs in methyltransferase-catalyzed modification of biomolecules. However, SAM and its analogs are expensive and unstable, degrading rapidly under physiological conditions. Thus, the availability of methods to prepare SAM in situ is desirable. In addition, synthetic methods to generate SAM analogs suffer from low yields and poor diastereoselectivity. The chlorinase SalL from the marine bacterium Salinispora tropica catalyzes the reversible, nucleophilic attack of chloride at the C5' ribosyl carbon of SAM leading to the formation of 5'-chloro-5'-deoxyadenosine (ClDA) with concomitant displacement of l-methionine. It has been demonstrated that the in vitro equilibrium of the SalL-catalyzed reaction favors the synthesis of SAM. In this chapter, we describe methods for the preparation of SalL, and the chemoenzymatic synthesis of SAM and SAM analogs from ClDA and l-methionine congeners using SalL. In addition, we describe procedures for the in situ chemoenzymatic synthesis of SAM coupled to DNA, peptide, and metabolite methylation, and to the incorporation of isotopes into alkylated products.
S-腺苷-L-甲硫氨酸（SAM）在生物学中普遍存在，是各种酶促反应中第二常见的辅因子。SAM的主要作用之一是核酸、蛋白质和代谢物的甲基化。甲基化通常赋予DNA和蛋白质调控功能，并导致特殊代谢物（如那些开发为药物的代谢物）的活性增加。人们对使用SAM类似物进行甲基转移酶催化的生物分子修饰越来越感兴趣。然而，SAM及其类似物价格昂贵且不稳定，在生理条件下会迅速降解。因此，原位制备SAM的方法是很有必要的。此外，合成SAM类似物的方法存在产率低和非对映选择性差的问题。来自海洋细菌热带盐孢菌的氯酶SalL催化氯离子对SAM的C5'核糖基碳进行可逆的亲核攻击，导致形成5'-氯-5'-脱氧腺苷（ClDA），同时置换L-甲硫氨酸。已经证明，SalL催化反应的体外平衡有利于SAM的合成。在本章中，我们描述了制备SalL的方法，以及使用SalL从ClDA和L-甲硫氨酸同系物化学酶促合成SAM和SAM类似物的方法。此外，我们还描述了与DNA、肽和代谢物甲基化以及将同位素掺入烷基化产物偶联的SAM原位化学酶促合成的程序。
