MhpA is a hydroxylase catalyzing the initial reaction of 3-(3-hydroxyphenyl)propionate catabolism in Escherichia coli K-12
MhpA is a hydroxylase catalyzing the initial reaction of 3-(3-hydroxyphenyl)propionate catabolism in Escherichia coli K-12
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MhpA 是一种羟化酶，可催化大肠杆菌 K-12 中 3-(3-羟苯基)丙酸分解代谢的初始反应
DOI：
10.1128/aem.02385-19
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发表时间：
2020
影响因子：
4.4
通讯作者：
Zhou Ning-Yi
中科院分区：
文献类型：
--
作者：
Xu Ying;Zhou Ning-Yi
据报道，大肠杆菌K-12和其他一些菌株能够利用3-（3-羟基苯基）丙酸酯（3HPP），这是木质素中苯丙酸酯的一种。虽然已经确定了参与3HPP分解代谢的其他酶及其降解物的相应基因，但催化其分解代谢第一步的3HPP 2-羟化酶尚未在生化和遗传水平上进行功能鉴定。在这项研究中，我们研究了大肠杆菌菌株K-12的MhpA（MhpA(K-12)）的功能和特性。基因缺失和互补表明mhpA对其在3HPP上的生长至关重要，但mhpA缺失菌株仍能在3-（2,3-二羟基苯基）丙酸酯（DHPP）上生长，DHPP是由3HPP转化为mhpA （K-12）的羟基化产物。MhpA（K-12）被过表达和纯化，它可能是一种聚合物，与大约等量摩尔的FAD紧密结合。使用NADH或NADPH作为辅助因子，纯化的MhpA（K-12）以类似的效率催化3HPP转化为DHPP。纯化的MhpA（K-12）通过高效液相色谱和液相色谱-质谱分析证实了3HPP转化为DHPP。生物信息学分析表明，MhpA（K-12）及其推测的同源物属于与功能鉴定的fad单加氧酶（羟化酶）在系统发育上距离较远的分类群。有趣的是，与相近的同源物相比，MhpA（K-12）在其C端大约多了150个残基，但其截断版本MhpA(K-12)（400）和MhpA(K-12)(480)（分别从C端删除了154和74个残基）都失去了活性。因此，MhpA（K-12）被证实是3HPP 2-羟化酶，催化3HPP转化为DHPP，这是3HPP降解的初始反应。苯丙酸酯及其羟基化衍生物在地球上普遍存在于木质素降解过程中。许多菌株具有在3HPP上生长的能力，3HPP是上述衍生物之一。羟基化被认为是其通过元途径进行有氧分解代谢的初始和重要步骤。我们研究的意义在于从大肠杆菌K-12中纯化的3HPP 2-羟化酶MhpA在生化和遗传水平上的功能鉴定和表征，因为该酶以前没有在任何细菌中从其编码基因表达，纯化和表征。这不仅填补了我们对微生物3HPP分解代谢关键步骤3HPP 2-羟化酶及其对应基因的理解空白，而且还提供了植物源苯丙酸及其羟化衍生物微生物降解多样性的另一个例子。
Escherichia coli K-12 and some other strains have been reported to be capable of utilizing 3-(3-hydroxyphenyl)propionate (3HPP), one of the phenylpropanoids from lignin. Although other enzymes involved in 3HPP catabolism and their corresponding genes from its degraders have been identified, 3HPP 2-hydroxylase, catalyzing the first step of its catabolism, has yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characteristics of MhpA from E. coli strain K-12 (MhpA(K-12)). Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA deletion strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpA(K-12). MhpA(K-12) was overexpressed and purified, and it was likely a polymer and tightly bound with an approximately equal number of moles of FAD. Using NADH or NADPH as a cofactor, purified MhpA(K-12) catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpA(K-12) was confirmed using highperformance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpA(K-12) and its putative homologues belonged to taxa that were phylogenetically distant from functionally identified FAD-containing monooxygenases (hydroxylases). Interestingly, MhpA(K-12) has approximately an extra 150 residues at its C terminus in comparison to its close homologues, but its truncated versions MhpA(K-12)(400) and MhpA(K-12)(480) (with 154 and 74 residues deleted from the C terminus, respectively) both lost their activities. Thus, MhpA(K-12) has been confirmed to be a 3HPP 2-hydroxylase catalyzing the conversion of 3HPP to DHPP, the initial reaction of 3HPP degradation. IMPORTANCE Phenylpropionate and its hydroxylated derivatives resulted from lignin degradation ubiquitously exist on the Earth. A number of bacterial strains have the ability to grow on 3HPP, one of the above derivatives. The hydroxylation was thought to be the initial and vital step for its aerobic catabolism via the meta pathway. The significance of our research is the functional identification and characterization of the purified 3HPP 2-hydroxylase MhpA from Escherichia colt K-12 at biochemical and genetic levels, since this enzyme has not previously been expressed from its encoding gene, purified, and characterized in any bacteria. It will not only fill a gap in our understanding of 3HPP 2-hydroxylase and its corresponding gene for the critical step in microbial 3HPP catabolism but also provide another example of the diversity of microbial degradation of plant-derived phenylpropionate and its hydroxylated derivatives.