Fu H, Alvarez M A, Khosla C, Bailey J E
Department of Chemical Engineering, Stanford University, California 94305-5025, USA.
Biochemistry. 1996 May 28;35(21):6527-32. doi: 10.1021/bi952957y.
TcmO is an O-methyltransferase that methylates the C-8 hydroxyl to Tcm B3, a four-ring aromatic intermediate in the tetracenomycin biosynthetic pathway of Streptomyces glaucescens. The gene encoding this enzyme was expressed in Streptomyces coelicolor CH999 together with the actinorhodin polyketide synthase (PKS) gene cluster, which is responsible for the biosynthesis of 3,8-dihydroxy-methylanthraquinone carboxylic acid (DMAC) and its decarboxylated analog, aloesaponarin. The resulting recombinant strain produced approximately equal quantities of aloesaponarin and a new product but no DMAC. Spectroscopic analysis revealed that the novel polyketide was the 3-O-methylated analog of DMAC. An in vitro radioisotopic assay was developed for tcmO. The enzyme requires S-adenosylmethionine as a co-substrate. It has a Km of 3 microM and a kcat of 2.7 min-1 for DMAC. A series of monocyclic, bicyclic, and tricyclic aromatic compounds were also tested as candidate substrates in vitro. Remarkably, none was modified by tcmO within detectable limits of the assay. Together, these results highlight the interesting molecular recognition features of this enzyme. On one hand, there appears to be some flexibility in the number and structures of unreactive rings, since both Tcm and B3 and DMAC are good substrates. However, 6-methylsalicylic acid, a monocyclic analog of the reactive ring, is not recognized by the enzyme. Likewise, neither aloesaponarin (which only differs from DMAC in the reactive ring) nor carminic acid (which only differs in the distal nonreactive ring) is modified. Thus, the binding energy for the tcmO-catalyzed methyl transfer appears to involve significant contributions from both the aromaticity and the functionality of polycyclic substrates.
TcmO是一种O-甲基转移酶，它将C-8羟基甲基化生成Tcm B3，Tcm B3是浅青紫链霉菌四环素生物合成途径中的一种四环芳香中间体。编码该酶的基因与放线紫红素聚酮合酶（PKS）基因簇一起在天蓝色链霉菌CH999中表达，放线紫红素聚酮合酶基因簇负责3,8-二羟基-甲基蒽醌羧酸（DMAC）及其脱羧类似物芦荟皂苷元的生物合成。所得重组菌株产生了大致等量的芦荟皂苷元和一种新产品，但没有产生DMAC。光谱分析表明，这种新型聚酮化合物是DMAC的3-O-甲基化类似物。开发了一种针对tcmO的体外放射性同位素测定法。该酶需要S-腺苷甲硫氨酸作为共底物。对于DMAC，它的Km为3 microM，kcat为2.7 min-1。一系列单环、双环和三环芳香化合物也在体外作为候选底物进行了测试。值得注意的是，在该测定的可检测限度内，没有一种被tcmO修饰。这些结果共同突出了这种酶有趣的分子识别特征。一方面，未反应环的数量和结构似乎存在一定的灵活性，因为Tcm B3和DMAC都是良好的底物。然而，6-甲基水杨酸，即反应环的单环类似物，不被该酶识别。同样，芦荟皂苷元（仅在反应环上与DMAC不同）和胭脂红酸（仅在远端非反应环上不同）都没有被修饰。因此，tcmO催化的甲基转移的结合能似乎涉及多环底物的芳香性和官能团的显著贡献。
