Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida.
Substrate specificity differences between two catechol 2,3-dioxygenases encoded by the TOL and NAH plasmids from Pseudomonas putida.
复制标题
由恶臭假单胞菌的 TOL 和 NAH 质粒编码的两种儿茶酚 2,3-双加氧酶之间的底物特异性差异。
DOI：
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
S. Harayama
中科院分区：
文献类型：
--
作者：
P. Cerdán;M. Rekik;S. Harayama
研究了两种儿茶酚2，3-双加氧酶的底物特异性，一种由TOL质粒pWW 0上的xylE编码，另一种由NAH 7质粒上的nahH编码。XylE儿茶酚2，3-双加氧酶催化儿茶酚、3-甲基儿茶酚和4-甲基儿茶酚的环断裂。NahH邻苯二酚2，3-双加氧酶在氧化3-甲基邻苯二酚时由于两种催化性质的缺陷而部分缺乏。首先，与XylE相比，NahH对3-甲基儿茶酚具有较低的kcat值，其次，NahH比XylE对3-甲基儿茶酚的自杀抑制更敏感。为了鉴定导致3-甲基儿茶酚氧化功效差异的XylE和NahH的氨基酸残基，测定了几种NahH-XylE杂合蛋白的3-甲基儿茶酚的kcat和kinact（自杀抑制的速率常数），每种杂合蛋白由N-末端区域的NahH序列和C-末端区域的XylE序列组成。结果表明，NahH序列中存在的单个氨基酸取代，His 250->Gln，是3-甲基儿茶酚kcat降低和kinact值增加的原因。除了在残基250处的取代之外，在残基77-102处的一些取代是NahH和XylE与3-甲基儿茶酚的kinact值的两倍差异的原因。我们还表明，3-甲基儿茶酚的自杀抑制的结合位点是不同的催化位点。
The substrate specificities of two catechol 2,3-dioxygenases, one encoded by xylE on the TOL plasmid pWW0 and the other encoded by nahH on the NAH7 plasmid, were investigated. The XylE catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol. The NahH catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties. First, NahH has a lower kcat value for 3-methylcatechol compared to XylE, and secondly, NahH is more susceptible than XylE to suicide inhibition by 3-methylcatechol. To identify the amino acid residues of XylE and NahH responsible for the differences in the efficacy of the 3-methylcatechol oxidation, kcat and kinact (the rate constant for suicide inhibition) for 3-methylcatechol were determined for several NahH-XylE hybrid proteins, each of which consisted of the NahH sequence in the N-terminal region and the XylE sequence in the C-terminal region. It is shown that a single amino acid substitution present in the NahH sequence, His250-->Gln, was responsible for the reduced kcat and increased kinact values for 3-methylcatechol. In addition to the substitution at residue 250, some substitution(s) at residues 77-102 were responsible for the twofold difference in the kinact values for NahH and XylE with 3-methylcatechol. We also show that the binding site of 3-methylcatechol for suicide inhibition is different from the catalytic site.
质粒NAH7的基因nahH的核苷酸序列和表达以及与TOL pWWO的基因xylE的同源性。
影响因子：
3.5
作者：
Ghosal,D;You,IS;Gunsalus,IC
通讯作者：
Gunsalus,IC