A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.
A mutagenic analysis of NahE, a hydratase-aldolase in the naphthalene degradative pathway.
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NahE（萘降解途径中的一种水合酶-醛缩酶）的诱变分析。
DOI：
10.1016/j.abb.2022.109471
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发表时间：
2023
影响因子：
3.9
通讯作者：
Whitman,ChristianP
中科院分区：
文献类型：
--
作者：
Lancaster,EmilyB;JohnsonJr,WilliamH;LeVieux,JakeA;Hardtke,HaleyA;Zhang,YanJessie;Whitman,ChristianP
NahE是一种水合酶醛缩酶，可将取代的反式苄基苯丙酮酸（H、OH或CO2 -）分别转化为苯甲醛、水杨醛或2-羧基苯甲醛和丙酮酸。萘是一种有毒的持久性环境污染物，该酶是一种细菌降解途径。序列、晶体学和诱变分析确定该酶是醛缩酶超家族中n -乙酰神经氨酸解酶（NAL）亚群的成员。因此，它具有一个保守的赖氨酸（Lys183）和酪氨酸（Tyr155），用于席夫碱的形成，以及一个GXXGE基序，用于结合丙酮酰羧酸基。钠he的硒代蛋氨酸衍生物的晶体结构显示了这些活性位点元素以及可能参与机制和/或特异性的附近残基。构建了5个活性位点氨基酸（Thr65、Trp128、Tyr155、Asn157和Asn281）的突变，并测量了动力学参数，以评估对催化的影响。结果表明，两个Trp128突变体（Phe和Tyr）对催化作用影响最小，而Thr65 （Val）和Asn281 （Leu）侧链较大的氨基酸对催化作用最大。将Tyr155转变为Phe和Asn157转变为Ala也会阻碍催化作用，其影响介于这两个极端之间。利用反应中间体的希夫碱的晶体结构，将这些观察结果置于结构背景中。底物、Na(CN)BH3、野生型酶和所选突变体的捕获实验与动力学分析基本一致，鉴定出两种水杨醛修饰赖氨酸：活性位点赖氨酸（Lys183）和活性位点外赖氨酸（Lys279）。后者可能是水杨醛抑制NahE的原因。总之，这些结果为nahe催化反应提供了新的见解。
NahE is a hydratase-aldolase that convertso-substitutedtrans-benzylidenepyruvates (H, OH, or CO2−) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is in a bacterial degradative pathway for naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of theN-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. A crystal structure of the selenomethionine derivative of NahE shows these active site elements along with nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on catalysis. The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. Changing Tyr155 to Phe and Asn157 to Ala also hinders catalysis, and the effects fall in between these extremes. These observations are put into a structural context using a crystal structure of the Schiff base of the reaction intermediate. Trapping experiments with substrate, Na(CN)BH3, and wild type enzyme and selected mutants mostly paralleled the kinetic analysis, and identified two salicylaldehyde-modified lysines: the active site lysine (Lys183) and one outside the active site (Lys279). The latter could be responsible for the observed inhibition of NahE by salicylaldehyde. Together, the results provide new insights into the NahE-catalyzed reaction.