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Uncommon Arrangement of Self-resistance Allows Biosynthesis of de novo Purine Biosynthesis Inhibitor that Acts as an Immunosuppressor
Journal of the American Chemical Society ( IF 14.4 ) Pub Date : 2023-12-05 , DOI: 10.1021/jacs.3c09600
Michio Sato 1 , Sakurako Sakano 1 , Miku Nakahara 1 , Yui Tamura 1 , Kodai Hara 1 , Hiroshi Hashimoto 1 , Yi Tang , Kenji Watanabe 1
Affiliation  

(−)-FR901483 (1) isolated from the fungus Cladobotryum sp. No.11231 achieves immunosuppression via nucleic acid biosynthesis inhibition rather than IL-2 production inhibition as accomplished by FK506 and cyclosporin A. Recently, we identified the frz gene cluster for the biosynthesis of 1. It contains frzK, a gene homologous to phosphoribosyl pyrophosphate amidotransferase (PPAT)that catalyzes the initial step of de novo purine biosynthesis. We speculated that frzK encodes a PPAT that escapes inhibition by 1 and functions as a self-resistance enzyme (SRE) for the producing host. Nevertheless, details remained elusive. Here, we report the biochemical and structural analyses of FrzK and its Escherichia coli counterpart, PurF. Recombinantly produced FrzK exhibited PPAT activity, albeit weaker than PurF, but evaded strong inhibition by 1. These results confirmed that the target of 1 is PPAT, and FrzK acts as an SRE by maintaining the de novo purine biosynthetic capability in the presence of 1. To understand how FrzK evades inhibition by 1, we determined the crystal structure of PurF in the complex with 1 and constructed a homology model of FrzK. Sequence and structural analyses of various PPATs identified that many residues unique to FrzK occur near the Flexible Loop that remains disordered when inactive but becomes ordered and covers up the active site upon activation by substrate binding. Kinetic characterizations of mutants of the unique residues revealed that the resistance of FrzK against 1 may be conferred by structurally predisposing the Flexible Loop to the active, closed conformation even in the presence of 1.

中文翻译:


罕见的自我抵抗排列允许从头生物合成嘌呤生物合成抑制剂,充当免疫抑制剂



(−)-FR901483 ( 1 ) 从真菌Cladobotryum sp. 中分离出来。 No.11231 通过核酸生物合成抑制而不是 FK506 和环孢菌素 A 实现的 IL-2 产生抑制来实现免疫抑制。最近,我们鉴定了1生物合成的frz基因簇。它含有frzK ,这是一种与磷酸核糖焦磷酸酰胺转移酶 (PPAT) 同源的基因,可催化嘌呤从头生物合成的第一步。我们推测frzK编码的 PPAT 可以逃脱1 的抑制,并作为生产宿主的自抗性酶 (SRE)。尽管如此,细节仍然难以捉摸。在这里,我们报告了 FrzK 及其大肠杆菌对应物 PurF 的生化和结构分析。重组产生的 FrzK 表现出 PPAT 活性,尽管比 PurF 弱,但避开了强抑制1 。这些结果证实1的靶标是PPAT,并且FrzK通过在1存在的情况下维持从头嘌呤生物合成能力而充当SRE。为了了解FrzK如何逃避1的抑制,我们确定了与1复合物中PurF的晶体结构,并构建了FrzK的同源模型。对各种 PPAT 的序列和结构分析发现,FrzK 特有的许多残基出现在柔性环附近,柔性环在不活动时保持无序,但在通过底物结合激活后变得有序并覆盖活性位点。 独特残基突变体的动力学表征表明,即使在1存在的情况下,FrzK 对1的抗性也可能是通过在结构上使柔性环倾向于活性、闭合构象而赋予的。
更新日期:2023-12-05
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