Hydrolytic polyketide shortening by Ayg1p, a novel enzyme involved in fungal melanin biosynthesis
Hydrolytic polyketide shortening by Ayg1p, a novel enzyme involved in fungal melanin biosynthesis
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DOI：
10.1074/jbc.m406758200
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发表时间：
2004-10-22
影响因子：
4.8
通讯作者：
Ebizuka, Y
中科院分区：
文献类型：
--
作者：
Fujii, I;Yasuoka, Y;Ebizuka, Y
pentaketide 1，3，6，8-tetrahydroxynaphthalene（T4 HN）是1，8-dihydroxynaphthalenemelanin的关键前体，是病原真菌的重要毒力因子，T4 HN被认为是pentaketide酶的直接产物。最近，我们发现了一种新的蛋白质，Ayg 1 p，在烟曲霉中从heptaketide前体YWA 1形成T4 HN的参与。为了研究其酶功能的机制，Ayg 1 p从过表达ayg 1基因的曲霉菌株中纯化。Ayg 1 p将萘并吡喃酮YWA 1转化为T4 HN，并释放出乙酰乙酸。虽然Ayg 1 p与已知酶没有显着的同源性，但其序列中存在丝氨酸蛋白酶型水解基序，并且丝氨酸特异性抑制剂强烈抑制活性。为了确定其催化残基，定点Ayg 1 p突变体在大肠杆菌中表达，并检测其酶活性。单取代突变S257 A、D352 A和H380 A导致Ayg 1 p酶活性完全丧失。这些结果表明催化三联体Asp（352）-His（380）-Ser（257）构成了Ayg 1 p的活性位点。从一个狄克逊图分析，2-乙酰基-1，3，6，8-四羟基萘被发现是一个强大的混合型抑制剂，表明参与的酰基酶中间体。这些研究支持了活性位点的Ser（257）作为亲核试剂攻击YWA 1侧链1 '-羰基并裂解萘环和侧链之间的碳-碳键的机制。乙酰乙酸随后通过水解从Ser（257）-O-乙酰乙酰化Ayg 1 p中释放出来。在黑色素生物合成中具有类似于Ayg 1 p活性的酶在任何其他生物体中尚未报道。
The pentaketide 1,3,6,8-tetrahydroxynaphthalene (T4HN) is a key precursor of 1,8-dihydroxynaphthalenemelanin, an important virulence factor in pathogenic fungi, where T4HN is believed to be the direct product of pentaketide synthases. We showed recently the involvement of a novel protein, Ayg1p, in the formation of T4HN from the heptaketide precursor YWA1 in Aspergillus fumigatus. To investigate the mechanism of its enzymatic function, Ayg1p was purified from an Aspergillus oryzae strain that overexpressed the ayg1 gene. The Ayg1p converted the naphthopyrone YWA1 to T4HN with a release of the acetoacetic acid. Although Ayg1p does not show significant homology with known enzymes, a serine protease-type hydrolytic motif is present in its sequence, and serine-specific inhibitors strongly inhibited the activity. To identify its catalytic residues, site-directed Ayg1p mutants were expressed in Escherichia coli, and their enzyme activities were examined. The single substitution mutations S257A, D352A, and H380A resulted in a complete loss of enzyme activity in Ayg1p. These results indicated that the catalytic triad Asp(352)-His(380)-Ser(257) constituted the active-site of Ayg1p. From a Dixon plot analysis, 2-acetyl-1,3,6,8-tetrahydroxynaphthalene was found to be a strong mixed-type inhibitor, suggesting the involvement of an acyl-enzyme intermediate. These studies support the mechanism in which the Ser(257) at the active site functions as a nucleophile to attack the YWA1 side-chain 1'-carbonyl and cleave the carbon-carbon bond between the naphthalene ring and the side chain. Acetoacetic acid is subsequently released from the Ser(257)-O-acetoacetylated Ayg1p by hydrolysis. An enzyme with activity similar to Ayg1p in melanin biosynthesis has not been reported in any other organism.