Structural Basis for the Formation of Acylalkylpyrones from Two β-Ketoacyl Units by the Fungal Type III Polyketide Synthase CsyB
Structural Basis for the Formation of Acylalkylpyrones from Two β-Ketoacyl Units by the Fungal Type III Polyketide Synthase CsyB
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DOI：
10.1074/jbc.m114.626416
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发表时间：
2015-02-20
影响因子：
4.8
通讯作者：
Abe, Ikuro
中科院分区：
文献类型：
--
作者：
Mori, Takahiro;Yang, Dengfeng;Abe, Ikuro
米曲霉的酰基吡喃酮合成酶CsyB催化乙酰乙酰-辅酶A、脂肪酰-辅酶A和丙二酰辅酶A一锅合成3-酰基-4-羟基-6-烷基-α-吡喃酮支架。这是第一个类型III型聚酮合成酶，它不仅执行聚酮链的延长，而且还执行两个β-酮酰基单元的缩合。分别在1.7埃、2.3埃和2.0埃分辨率下对野生型CsyB及其突变体I375F和I375W的晶体结构进行了解析。晶体结构揭示了一个独特的活性中心结构，除了传统的带有Cys-His-ASN催化三联体的延伸/环化口袋和用于结合脂肪酰链的长疏水隧道外，还具有一个迄今未知的用于容纳乙酰乙酰-CoA起始剂的新型口袋。这些结构还表明存在一个可能的亲核水分子，该分子被位于活性中心的His-377和Cys-155的氢键网络激活。此外，体外酶反应证实，(H2O)-O-18分子的O-18原子在酶作用下被整合到最终产物中。这些观察结果表明，酶反应是通过将乙酰乙酰-辅酶A负载到Cys-155上而启动的，随后亲核水断裂硫酯键生成β-酮酸中间体，该中间体被放置在新的口袋中。然后，脂肪酰辅酶A与一个丙二酰辅酶A分子通过聚酮链延伸生成第二个β-酮酰基单元，并与β-酮酸缩合生成最终产品。事实上，基于结构的I375F和I375W突变对新口袋的立体调制导致了底物链长的特异性改变。
The acylalkylpyrone synthase CsyB from Aspergillus oryzae catalyzes the one-pot formation of the 3-acyl-4-hydroxy-6-alkyl-alpha-pyrone scaffold from acetoacetyl-CoA, fatty acyl-CoA, and malonyl-CoA. This is the first type III polyketide synthase that performs not only the polyketide chain elongation but also the condensation of two beta-ketoacyl units. The crystal structures of wild-type CsyB and its I375F and I375W mutants were solved at 1.7-, 2.3-, and 2.0-angstrom resolutions, respectively. The crystal structures revealed a unique active site architecture featuring a hitherto unidentified novel pocket for accommodation of the acetoacetyl-CoA starter in addition to the conventional elongation/cyclization pocket with the Cys-His-Asn catalytic triad and the long hydrophobic tunnel for binding the fatty acyl chain. The structures also indicated the presence of a putative nucleophilic water molecule activated by the hydrogen bond networks with His-377 and Cys-155 at the active site center. Furthermore, an in vitro enzyme reaction confirmed that the O-18 atom of the (H2O)-O-18 molecule is enzymatically incorporated into the final product. These observations suggested that the enzyme reaction is initiated by the loading of acetoacetyl-CoA onto Cys-155, and subsequent thioester bond cleavage by the nucleophilic water generates the beta-keto acid intermediate, which is placed within the novel pocket. The second beta-ketoacyl unit is then produced by polyketide chain elongation of fatty acyl-CoA with one molecule of malonyl-CoA, and the condensation with the beta-keto acid generates the final products. Indeed, steric modulation of the novel pocket by the structure-based I375F and I375W mutations resulted in altered specificities for the chain lengths of the substrates.