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首页> 外文期刊>Journal of Molecular Biology >Enhanced specificity of mint geranyl pyrophosphate synthase by modifying the R-loop interactions.
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Enhanced specificity of mint geranyl pyrophosphate synthase by modifying the R-loop interactions.

机译：通过修饰R环相互作用增强了薄荷香叶基焦磷酸合成酶的特异性。

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Isoprenoids, most of them synthesized by prenyltransferases (PTSs), are a class of important biologically active compounds with diverse functions. The mint geranyl pyrophosphate synthase (GPPS) is a heterotetramer composed of two LSU.SSU (large/small subunit) dimers. In addition to C(10)-GPP, the enzyme also produces geranylgeranyl pyrophosphate (C(20)-GGPP) in vitro, probably because of the conserved active-site structures between the LSU of mint GPPS and the homodimeric GGPP synthase from mustard. By contrast, the SSU lacks the conserved aspartate-rich motifs for catalysis. A major active-site cavity loop in the LSU and other trans-type PTSs is replaced by the regulatory R-loop in the SSU. Only C(10)-GPP, but not C(20)-GGPP, was produced when intersubunit interactions of the R-loop were disrupted by either deletion or multiple point mutations. The structure of the deletion mutant, determined in two different crystal forms, shows an intact (LSU.SSU)(2) heterotetramer, as previously observed in the wild-type enzyme. The active-site of LSU remains largely unaltered, except being slightly more open to the bulk solvent. The R-loop of SSU acts by regulating the product release from LSU, just as does its equivalent loop in a homodimeric PTS, which prevents the early reaction intermediates from escaping the active site of the other subunit. In this way, the product-retaining function of R-loop provides a more stringent control for chain-length determination, complementary to the well-established molecular ruler mechanism. We conclude that the R-loop may be used not only to conserve the GPPS activity but also to produce portions of C(20)-GGPP in mint.
机译：类异戊二烯是大多数重要的具有多种功能的生物活性化合物，它们大多由异戊二烯基转移酶（PTS）合成。薄荷香叶基焦磷酸合酶（GPPS）是由两个LSU.SSU（大/小亚基）二聚体组成的异四聚体。除C（10）-GPP外，该酶还可以在体外产生香叶基香叶基香叶基磷酸（C（20）-GGPP），这可能是由于薄荷GPPS的LSU与芥菜的同型二聚体GGPP合酶之间存在保守的活性位点结构。相反，SSU缺乏保守的富含天冬氨酸的催化基序。 LSU和其他反型PTS中的主要活动部位腔环被SSU中的调节性R环取代。当R环的亚基相互作用被缺失或多点突变破坏时，仅产生C（10）-GPP，而不产生C（20）-GGPP。如先前在野生型酶中观察到的，以两种不同的晶体形式确定的缺失突变体的结构显示出完整的（LSU.SSU）（2）异四聚体。 LSU的活性部位基本保持不变，但对主体溶剂的开放程度略高。 SSU的R环通过调节LSU的产物释放来发挥作用，就像它在同型二聚体PTS中的等效环一样，它可以防止早期的反应中间体逸出另一个亚基的活性位点。这样，R环的产物保留功能为链长确定提供了更严格的控制，与完善的分子标尺机制互补。我们得出的结论是，R环不仅可以用于保存GPPS活性，而且可以在薄荷糖中产生C（20）-GGPP的一部分。

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