SAM自由基酶研究进展:新反应与新机制

Recent progress in radical SAM enzymes:New reactions and mechanisms

S-腺苷-L-蛋氨酸(S-adenosyl-L-methionine,SAM)自由基酶是当今酶学领域的研究热点.该类酶通过结合的辅因子SAM和[4Fe-4S]簇催化生物体中一系列重要的自由基反应,自2001年被正式命名以来,成员不断壮大,目前已成为最大的酶家族之一.近年来,SAM自由基酶领域有大量新反应和新催化机制被报道.本文对近5年部分代表性成果进行酶催化机制的介绍,内容涉及核糖体肽翻译后修饰、核苷类化合物以及多种小分子生物合成.通过底物分类,让读者更容易理解SAM自由基酶催化反应的广泛性与多样性.同时对该领域新发现的新颖的有机金属催化自由基反应机制进行了介绍,并对SAM自由基酶领域的未来发展方向进行展望.

S-adenosyl-L-methionine(SAM)is the second largest cofactor in the human body,and its metabolism is closely related to various physiological activities.In addition to a methyl donor,SAM is also used by radical SAM enzymes with a[4Fe-4S]cluster to catalyze a series of radical reactions.Since the discovery of this enzyme family,it has been found to play important physiological functions in all kingdoms of life.According to bioinformatics prediction,over 220000 radical SAM enzymes are involved in more than 85 biochemical transformations.Almost all radical SAM enzymes have a conserved Cxxx Cxx C motif,which coordinates the[4Fe-4S]cluster.The reduced[4Fe-4S]cluster provides an electron to SAM to cleave the C;-S bond of SAM and generates a 5′-deoxyadenosine radical(5′-d A·).This radical then grabs a hydrogen atom from the substrate to generate the substrate radical and initiates many different types of reactions.These include thioether crosslinking reactions,radical addition reactions that generate C–C bonds,aliphatic etherification that generates C-O bonds,oxidation reactions,complex rearrangement reactions,methylation,methylthiolation,cyclopropanation reactions and so on.The nonclassical radical SAM enzyme Dph2 cleaves the C;–S bond of SAM to generate a3-amino-3-carboxylpropyl radical(ACP radical),which is added to a histidine residue of the substrate protein elongation factor EF2 for diphthamide biosynthesis.Radical SAM enzymes have a very rich substrate scope,including ribosomally synthesized and posttranslationally modified peptides(Ri PPs),proteins,nucleosides and all kinds of small molecules.Classifying by the type of substrate,this review summarizes some of the newly discovered radical SAM enzymes since 2015 and introduces the catalytic mechanism.In addition to new enzymes and reactions,strides have been made in mechanistic studies of radical SAM enzymes in the past several years.Using rapid freeze-quench(RFQ)combined with electron spin resonance spectroscopy(EPR)and electron-nucleus double resonance spectroscopy(ENDOR),researchers have captured and characterized two types of novel intermediates in radical SAM enzymes.These two intermediates are organometallic species with Fe–C bonds formed by the iron-sulfur cluster with the 5′-d A radical and ACP radical,respectively.These findings answer the long-standing question of how enzymes control active organic radical species in radical SAM enzymes and the regioselectivity of SAM cleavage.Finally,we look forward to the future directions of radical SAM enzymes.As discussed above,radical SAM enzymes catalyze numerous difficult reactions,such as C-H activation and C-C bond formation.New enzymatic reactions with unnatural substrates could be developed with protein engineering.Therefore,natural product analogs could be easily made for drug development.Furthermore,in the radical SAM chemistry,only Dph2 can cleave SAM unconventionally to generate the ACP radical,other than the 5′-d A radical from all the other radical SAM enzymes.Whether there are more enzymes using this mechanism to catalyze the reaction remains unexplored.We expect that more Dph2-like enzymes that can generate ACP radicals will be discovered in the future.Finally,the drawback for almost all the reported radical SAM enzymes thus far is their low efficiency.Protein engineering or other strategies that could increase the stability and efficiency of radical SAM enzymes are in high demand.Only by this means would we expect more widespread use of radical SAM enzymes as biocatalysts in synthetic biology.