HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866
HipH Catalyzes the Hydroxylation of 4-Hydroxyisophthalate to Protocatechuate in 2,4-Xylenol Catabolism by Pseudomonas putida NCIMB 9866
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HipH 在恶臭假单胞菌 NCIMB 9866 的 2,4-二甲苯酚分解代谢中催化 4-羟基间苯二甲酸酯羟基化为原儿茶酸酯
DOI：
10.1128/aem.03105-15
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发表时间：
2016-01-01
影响因子：
4.4
通讯作者：
Zhou, Ning-Yi
中科院分区：
文献类型：
--
作者：
Chao, Hong-Jun;Chen, Yan-Fei;Zhou, Ning-Yi
摘要 除了在对甲酚上生长外，恶臭假单胞菌 NCIMB 9866 是唯一报道的能够在 2,4-二甲酚上有氧生长的菌株，2,4-二甲酚被美国环境保护局列为优先污染物。先前已报道了参与对甲基氧化的几种酶以及相应的基因。催化分解代谢中间体 4-羟基间苯二甲酸酯氧化成环裂解底物原儿茶酸的酶也从菌株 NCIMB 9866 中纯化，但其遗传决定子仍然不可用。在这项研究中，通过转座子诱变从菌株NCIMB 9866中克隆了编码4-羟基间苯二甲酸羟化酶的基因hipH。纯化的重组 HipH-His6 被发现是一种二聚体蛋白，分子量约为 110 kDa。 HipH-His6 催化 4-羟基间苯二甲酸酯羟基化为原儿茶酸，比活性为 1.54 U mg−1，4-羟基间苯二甲酸酯与 NADPH 的表观 Km 值为 11.40 ± 3.05 μM，与 NADH 的表观 Km 值为 11.23 ± 2.43 μM，NADPH 和 NADH 的表观 Km 值相似 （分别为 64.31 ± 13.16 和 72.76 ± 12.06 μM）。 HipH-His6 4-羟基间苯二甲酸酯羟基化生成的原儿茶酸的身份也已通过高效液相色谱和质谱法得到证实。基因转录分析、基因敲除和互补表明，hipH 对于该菌株的 2,4-二甲苯酚分解代谢至关重要，但对于对甲酚分解代谢则不然。这填补了我们对编码 2,4-二甲酚分解代谢关键步骤的基因理解的空白，并且还提供了结构相似化合物的微生物分解代谢的生化和遗传多样性的另一个例子。
ABSTRACT In addition to growing on p-cresol, Pseudomonas putida NCIMB 9866 is the only reported strain capable of aerobically growing on 2,4-xylenol, which is listed as a priority pollutant by the U.S. Environmental Protection Agency. Several enzymes involved in the oxidation of the para-methyl group, as well as the corresponding genes, have previously been reported. The enzyme catalyzing oxidation of the catabolic intermediate 4-hydroxyisophthalate to the ring cleavage substrate protocatechuate was also purified from strain NCIMB 9866, but its genetic determinant is still unavailable. In this study, the gene hipH, encoding 4-hydroxyisophthalate hydroxylase, from strain NCIMB 9866 was cloned by transposon mutagenesis. Purified recombinant HipH-His6 was found to be a dimer protein with a molecular mass of approximately 110 kDa. HipH-His6 catalyzed the hydroxylation of 4-hydroxyisophthalate to protocatechuate with a specific activity of 1.54 U mg−1 and showed apparent Km values of 11.40 ± 3.05 μM for 4-hydroxyisophthalate with NADPH and 11.23 ± 2.43 μM with NADH and similar Km values for NADPH and NADH (64.31 ± 13.16 and 72.76 ± 12.06 μM, respectively). The identity of protocatechuate generated from 4-hydroxyisophthalate hydroxylation by HipH-His6 has also been confirmed by high-performance liquid chromatography and mass spectrometry. Gene transcriptional analysis, gene knockout, and complementation indicated that hipH is essential for 2,4-xylenol catabolism but not for p-cresol catabolism in this strain. This fills a gap in our understanding of the gene that encodes a critical step in 2,4-xylenol catabolism and also provides another example of biochemical and genetic diversity of microbial catabolism of structurally similar compounds.