J Craig Venter Institute, 9704 Rockville, MD 20850, USA.
BMC Genomics. 2011 Jan 11;12:21. doi: 10.1186/1471-2164-12-21.
Enzymes in the radical SAM (rSAM) domain family serve in a wide variety of biological processes, including RNA modification, enzyme activation, bacteriocin core peptide maturation, and cofactor biosynthesis. Evolutionary pressures and relationships to other cellular constituents impose recognizable grammars on each class of rSAM-containing system, shaping patterns in results obtained through various comparative genomics analyses.
An uncharacterized gene cluster found in many Actinobacteria and sporadically in Firmicutes, Chloroflexi, Deltaproteobacteria, and one Archaeal plasmid contains a PqqE-like rSAM protein family that includes Rv0693 from Mycobacterium tuberculosis. Members occur clustered with a strikingly well-conserved small polypeptide we designate "mycofactocin," similar in size to bacteriocins and PqqA, precursor of pyrroloquinoline quinone (PQQ). Partial Phylogenetic Profiling (PPP) based on the distribution of these markers identifies the mycofactocin cluster, but also a second tier of high-scoring proteins. This tier, strikingly, is filled with up to thirty-one members per genome from three variant subfamilies that occur, one each, in three unrelated classes of nicotinoproteins. The pattern suggests these variant enzymes require not only NAD(P), but also the novel gene cluster. Further study was conducted using SIMBAL, a PPP-like tool, to search these nicotinoproteins for subsequences best correlated across multiple genomes to the presence of mycofactocin. For both the short chain dehydrogenase/reductase (SDR) and iron-containing dehydrogenase families, aligning SIMBAL's top-scoring sequences to homologous solved crystal structures shows signals centered over NAD(P)-binding sites rather than over substrate-binding or active site residues. Previous studies on some of these proteins have revealed a non-exchangeable NAD cofactor, such that enzymatic activity in vitro requires an artificial electron acceptor such as N,N-dimethyl-4-nitrosoaniline (NDMA) for the enzyme to cycle.
Taken together, these findings suggest that the mycofactocin precursor is modified by the Rv0693 family rSAM protein and other enzymes in its cluster. It becomes an electron carrier molecule that serves in vivo as NDMA and other artificial electron acceptors do in vitro. Subclasses from three different nicotinoprotein families show "only-if" relationships to mycofactocin because they require its presence. This framework suggests a segregated redox pool in which mycofactocin mediates communication among enzymes with non-exchangeable cofactors.
radical SAM（rSAM）结构域家族中的酶在多种生物过程中发挥作用，包括 RNA 修饰、酶激活、细菌素核心肽成熟和辅因子生物合成。进化压力和与其他细胞成分的关系对每一类含有 rSAM 的系统施加了可识别的语法，从而在通过各种比较基因组学分析获得的结果中形成模式。
在许多放线菌中发现的一个未被描述的基因簇，以及偶尔在厚壁菌门、绿弯菌门、δ变形菌门和一个古菌质粒中发现的该基因簇，含有一个类似于 PqqE 的 rSAM 蛋白家族，其中包括结核分枝杆菌的 Rv0693。这些成员与一个惊人地保守的小多肽簇集在一起，我们将其命名为“mycofactocin”，其大小与细菌素和 PqqA 相似，后者是吡咯喹啉醌（PQQ）的前体。基于这些标记物分布的部分系统发育轮廓分析（Phylogenetic Profiling，PPP）不仅可以识别 mycofactocin 簇，还可以识别出第二个得分较高的蛋白质层。令人惊讶的是，这个层次充满了每个基因组多达 31 个成员，这些成员来自三个不同的烟酰胺蛋白家族的变体亚家族，每个家族都存在于三个不相关的烟酰胺蛋白家族中。这种模式表明，这些变体酶不仅需要 NAD(P)，还需要新型基因簇。进一步的研究使用 SIMBAL，一种类似于 PPP 的工具，在这些烟酰胺蛋白中搜索与 mycofactocin 存在最相关的多个基因组的子序列。对于短链脱氢酶/还原酶（SDR）和含铁脱氢酶家族，将 SIMBAL 得分最高的序列与同源已解决的晶体结构对齐，显示信号集中在 NAD(P)-结合位点上，而不是在底物结合或活性位点残基上。对其中一些蛋白质的先前研究表明存在不可交换的 NAD 辅助因子，因此体外酶的活性需要人工电子受体，如 N,N-二甲基-4-亚硝基苯胺（NDMA）才能使酶循环。
综上所述，这些发现表明 mycofactocin 前体由 Rv0693 家族 rSAM 蛋白和其簇中的其他酶修饰而成。它成为一种电子载体分子，在体内充当 NDMA 和其他人工电子受体在体外的作用。来自三个不同烟酰胺蛋白家族的亚类与 mycofactocin 呈“若非有则无”的关系，因为它们需要 mycofactocin 的存在。这个框架表明存在一个分隔的氧化还原池，其中 mycofactocin 在具有不可交换辅助因子的酶之间进行通讯。
