Mutational Analysis of the C-C Bond Cleaving Enzyme Phloretin Hydrolase from Eubacterium ramulus
Mutational Analysis of the C-C Bond Cleaving Enzyme Phloretin Hydrolase from Eubacterium ramulus
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DOI：
10.1007/s11244-013-0196-x
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发表时间：
2014-03-01
影响因子：
3.6
通讯作者：
Grogan, Gideon
中科院分区：
文献类型：
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作者：
Frank, Annika;Siirola, Elina;Grogan, Gideon
来自枝真杆菌（Eubacterium ramulus，Phy）的根皮素水解酶催化二氢查耳酮根皮素水解成间苯三酚和根皮酸，在其底物上进行正式的逆傅克酰化反应。其最接近的序列同源物（具有25%氨基酸序列同一性）是来自荧光假单胞菌的二乙酰间苯三酚水解酶（Phlg），其催化其底物的类似水解脱酰。已经确定了Phlg的结构并提出了催化机理（J Biol Chem 285：4603-4611，2010）。为了比较Phy与Phlg的催化特性，表达了编码Phy的基因，并纯化和结晶了酶。X射线荧光扫描识别出晶体中的锌。Phy的同源性模型，基于Phlg（PDB代码3 HWP）的结构，告知了酶的点突变体文库的构建，靶向与Phlg共享的残基，这些残基被认为参与锌结合和底物的芳环上的酰基和苯酚官能团的识别。His 123、His 251、Glu 154和Glu 255（保守的锌结合残基）的突变导致表达差或活性大大降低的变体; Tyr 115和His 203的突变（被认为分别在间苯三酚环的1位和3位结合苯酚基团）导致活性降低15倍的变体和无活性变体。这些结果表明，在某些方面的保护机制和底物识别之间的Phy和Phlg，和锌依赖性的C-C水解酶的这种类型的催化特性一般。
Phloretin hydrolase from Eubacterium ramulus (Phy) catalyzes the hydrolysis of the dihydrochalcone phloretin to phloroglucinol and phloretic acid, performing a formal retro- Friedel-Crafts acylation reaction on its substrate. Its closest sequence homolog, of 25 % amino acid sequence identity, is diacetyl phloroglucinol hydrolase (Phlg) from Pseudomonas fluorescens, which catalyses a similar, hydrolytic, de-acylation of its substrate. The structure of Phlg has been determined and a catalytic mechanism proposed (J Biol Chem 285:4603-4611, 2010). In order to compare the catalytic characteristics of Phy with Phlg, the gene encoding Phy was expressed and the enzyme purified and crystallised. An X-ray fluorescence scan identified zinc within the crystals. A homology model of Phy, based on the structure of Phlg (PDB code 3HWP), informed the construction of a point mutant library of the enzyme, targeting residues shared with Phlg that are thought to be involved in zinc binding and the recognition of acyl and phenol functionality on the aromatic ring of the substrates. Mutation of His123, His251, Glu154 and Glu255 (conserved zinc binding residues) resulted in variants that were either poorly expressed, or of much reduced activity; Mutation of Tyr115 and His203, thought to bind the phenol groups in the 1-and 3-positions of the phloroglucinol ring respectively, resulted in variants of 15-fold reduced activity and an inactive variant. These results are suggestive of conservation of some aspects of mechanism and substrate recognition between Phy and Phlg, and of the catalytic characteristics of Zn-dependent C-C hydrolases of this type in general.