S-Adenosyl-L-methionine:hydroxide adenosyltransferase:: A SAM enzyme
S-Adenosyl-L-methionine:hydroxide adenosyltransferase:: A SAM enzyme
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DOI：
10.1002/anie.200800794
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发表时间：
2008-01-01
影响因子：
16.6
通讯作者：
O'Hagan, David
中科院分区：
文献类型：
--
作者：
Deng, Hai;Botting, Catherine H.;O'Hagan, David
S-腺苷-L-蛋氨酸(SAM，1)在酶学中具有多种作用。它最常参与的是甲基转移反应，在此反应中，它将其甲基转移到O、N、S和各种底物的催化剂上。SAM还在不饱和脂肪酸的环丙烷化反应中贡献一个亚甲基。[2]有一种SAM脱羧酶，[3]它启动多胺的生物合成，也许最奇怪的是，SAM是至少三种铁硫酶中5‘-脱氧腺苷的来源。[4]SAM是几种代谢物生物合成的前体，包括植物中的乙烯，[5]生物素和环氧奎宁。[6]氟化[7]和氯化[8]酶已被报道利用SAM作为底物；在每种情况下，卤离子都介导SAM(1)5‘-位的亲核攻击，以生成相应的5’-卤代-5‘-脱氧腺苷(5’XDA)产物2或3和L-蛋氨酸(L-蛋氨酸；图式1)。这些反应，与SAM依赖的甲基转移酶，[1]是为数不多的已知的酶促SN2型反应。[9，10]用来自基因组测序的数据检查氟化酶和氯化酶氨基酸序列，发现与DUF62超家族(图1；DUF=功能未知的结构域)的同源性(<；36%)。[11]DUF62蛋白没有被赋予任何功能。这些基因的分布是有限的；一般来说，它们只存在于极端微生物和病原体相关微生物中。在结构蛋白质组学筛选程序中，来自四个不同嗜极菌的四个不同的duf62基因的蛋白质产物已经成为过表达和X射线晶体结构评估的对象。[12]四个duf62蛋白结构[12]彼此几乎相同，并且几乎可以重叠在先前报道的氟化酶[10]和氯化酶[8]结构上。其中一种酶来自P.horikoshii OT3，一种最适生长温度为988℃的微生物，它是在1395米深的日本太平洋冲绳海槽喷口分离出来的。[13]对结构的检查显示，腺苷分子配位在与氟化酶活性部位相对应的亚基位置(图2)。氟化酶和氯化酶也与腺苷共结晶，并且在重新提纯的horikoshii结构中的腺苷分子几乎可以与氟化酶和氯化物酶结构中的腺苷分子重叠。例如，天冬氨酸羧酸(Asp7)锚定腺苷核糖部分的2‘-和3’-OH基团。在此背景下，我们将Horikoshii OT3的DUF62基因重新克隆到大肠杆菌中，并进行了高效表达和纯化。[14]该蛋白在高浓度(>；10 mM)的卤离子中孵育后不能与SAM发生氟化或氯化反应。[15]然而，与亲核卤化物反应(方案2)类似，该酶可以通过SAM(1)的C5‘处的氢氧化物离子的攻击催化SAM转化为腺苷(4)。
S-Adenosyl-L-methionine (SAM, 1) has a variety of roles in enzymology. It is most commonly involved in methyl-transfer reactions,[1] in which it transfers its methyl group to O, N, S, and Catoms of various substrates. SAM also donates a methylene group in the cyclopropanation of unsaturated fatty acids.[2] There is a SAM decarboxylase,[3] which initiates polyamine biosynthesis, and, perhaps most exotically, SAM is the source of 5’-deoxyadenosyl radicals in at least three iron–sulfur enzymes.[4] SAM acts as a precursor in the biosynthesis of several metabolites, including ethylene in plants,[5] biotin, and epoxyqueuosine.[6] Fluorination [7] and chlorination [8] enzymes have been reported that utilize SAM as a substrate; in each case, the halide ion mediates a nucleophilic attack at the 5’-position of SAM (1) to generate the corresponding 5’-halo-5’-deoxyadenosine (5’XDA) product 2 or 3 and L-methionine (L-Met; Scheme1). These reactions, withSAM-dependent methyl transferases,[1] are among the few enzymatic SN2-type reactions known.[9, 10] Examination of the fluorinase and chlorinase amino acid sequences with data derived from genome sequencing reveals an identity (< 36%) to the DUF62 superfamily (Figure 1; DUF= domain of unknown function).[11] No function has been assigned to the DUF62 proteins. The distribution of these genes is restricted; in general they are found only in extremophile and pathogen-related microorganisms. The protein products of four different duf62 genes from four different extremophiles have been the subject of overexpression and X-ray crystal-structure evaluation in structuralproteomics screening programs.[12] The four DUF62 protein structures [12] are nearly identical with each other and almost superimposable on the previously reported fluorinase [10] and chlorinase [8] structures. One of these enzymes is from P. horikoshii OT3, a microorganism with an optimum growth temperature of 988C, which was isolated at a depth of 1395 m from an Okinawa trough vent in the Japanese Pacific Ocean.[13] Examination of the structure reveals an adenosine molecule coordinated at an intersubunit position corresponding to the fluorinase active site (Figure 2). The fluorinase and chlorinase also cocrystallize with adenosine, and the adenosine molecule in the re-refined P. horikoshii structure is almost superimposable on that in the structures of the fluorinase and the chlorinase. For example, an aspartate carboxylate (Asp7) anchors the 2’-and 3’-OH groups of the adenosine ribose moiety. With this background, we recloned the DUF62 gene of P. horikoshii OT3 into E. coli and overexpressed and purified the protein.[14] The protein was unable to mediate a fluorination or chlorination reaction with SAM after incubation at high concentrations (> 10 mm) of halide ions.[15] However, a novel activity was apparent in that the enzyme could catalyze the conversion of SAM into adenosine (4) by the attack of a hydroxide ion (from water) at C5’of SAM (1), in analogy with nucleophilic halide reactions (Scheme 2).