Jacobsen J R, Cane D E, Khosla C
Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
Biochemistry. 1998 Apr 7;37(14):4928-34. doi: 10.1021/bi9729920.
Modular polyketide synthases such as 6-deoxyerythronolide B synthase (DEBS) catalyze the biosynthesis of structurally complex natural products by repetitive condensation of simple carboxylic acid monomers. The synthase can be divided into groups of domains, called "modules", each of which is responsible for one cycle of chain extension and processing. The modular nature of these enzymes suggests that the biosynthetic pathway might be rationally reprogrammed by manipulation of synthases at the domain level. Although, several examples of successful engineering of DEBS have been reported, a critical issue which has not been well-studied is the tolerance of "downstream" active sites to nonnatural substrates. Here, we report that the terminal modules of DEBS, which normally process highly functionalized intermediates, are competent to carry out their natural functions on smaller, more simple substrates. Expressed in the absence of other DEBS proteins, the DEBS3 protein, which normally carries out the final two extension cycles in the synthesis of 6-deoxyerythronolide B (6-dEB), is spontaneously primed with a C3 carboxylic acid. This substrate is then extended through two condensation cycles to form a triketide. Tolerance of the "shortened" intermediates in the biosynthesis of this triketide, in combination with results reported elsewhere [Jacobsen, J. R., Hutchinson, C. R., Cane, D. E., and Khosla, C. (1997) Science 277, 367-369], suggests that relaxed substrate specificity may be a common feature of modular polyketide synthases. Interestingly, priming of DEBS3 appears to proceed, not by acyltransfer from propionyl-CoA, but by decarboxylation of an enzyme-bound methylmalonyl extender unit. This is the second example of decarboxylative priming within DEBS [see also Pieper, R., Gokhale, R. S., Luo, G., Cane, D. E., and Khosla, C. (1997) Biochemistry 36, 1846-1851] and suggests that, in the absence of an acceptable primer (or transferred intermediate), decarboxylative priming of ketosynthase domains may be a general property of modular polyketide synthases.
模块化聚酮合酶，如6-脱氧红霉内酯B合酶（DEBS），通过简单羧酸单体的重复缩合反应催化结构复杂的天然产物的生物合成。该合酶可分为若干组结构域，称为“模块”，每个模块负责一个链延伸和加工循环。这些酶的模块化性质表明，通过在结构域水平上对合酶进行操作，生物合成途径可能会被合理地重新编程。尽管已经报道了几个成功改造DEBS的例子，但一个尚未得到充分研究的关键问题是“下游”活性位点对非天然底物的耐受性。在这里，我们报道DEBS的末端模块，其通常处理高度官能化的中间体，能够在更小、更简单的底物上执行其天然功能。在没有其他DEBS蛋白的情况下表达时，通常在6-脱氧红霉内酯B（6-dEB）合成中执行最后两个延伸循环的DEBS3蛋白会自发地以C3羧酸为起始物。然后该底物通过两个缩合循环进行延伸，形成一个三酮化合物。在该三酮化合物生物合成中对“缩短”中间体的耐受性，与其他地方报道的结果[雅各布森，J.R.，哈钦森，C.R.，凯恩，D.E.，和科斯拉，C.（1997年）《科学》277，367 - 369]相结合，表明宽松的底物特异性可能是模块化聚酮合酶的一个共同特征。有趣的是，DEBS3的起始似乎不是通过丙酰辅酶A的酰基转移，而是通过酶结合的甲基丙二酰延伸单元的脱羧作用。这是DEBS内脱羧起始的第二个例子[另见皮珀，R.，戈卡尔，R.S.，罗，G.，凯恩，D.E.，和科斯拉，C.（1997年）《生物化学》36，1846 - 1851]，并表明在没有可接受的起始物（或转移中间体）的情况下，酮合酶结构域的脱羧起始可能是模块化聚酮合酶的一个普遍特性。
