The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β-cyano-L-alanine hydratase/nitrilase
The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode β-cyano-L-alanine hydratase/nitrilase
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DOI：
10.1074/jbc.m007890200
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发表时间：
2001-01-26
影响因子：
4.8
通讯作者：
Weiler, EW
中科院分区：
文献类型：
--
作者：
Piotrowski, M;Schönfelder, S;Weiler, EW
腈酶（Nitrilases, EC 3.5.5.1）是一种催化腈水解成相应碳酸的酶。在拟南芥已知的4种硝化酶中，同种异构体NIT4是差异最大的一种，在非芸苔科植物如烟草和水稻中也发现了NITI的同源物。我们将拟南芥NIT4作为六组氨酸标签融合蛋白在大肠杆菌中表达。纯化后的酶对高等植物中氰化物解毒的中间产物- p -氰- l -丙氨酸（Ala(CN)）具有很强的底物特异性。有趣的是，不仅天冬氨酸和天冬酰胺被鉴定为NIT4催化Ala（CN）水解的产物，Asn本身不是NIT4的底物，这表明它不是中间产物，而是两种反应产物之一。因此，NIT4同时具有腈酶和腈水合酶活性。多项证据表明，这两种反应的催化中心是相同的。烟叶N. NIT4同源物也催化相同的反应，烟叶A. thaliana、N. tabacum和Lupinus angustifolius的蛋白提取物也在体外将Ala（CN）转化为Asp和Asn。在乙烯生物合成过程中，硝酸氮可能在氰化物解毒过程中发挥作用，因为拟蓝衰老叶片提取物的Ala（CN）水合酶/硝化酶活性高于非衰老组织提取物。
Nitrilases (nitrile aminohydrolases, EC 3.5.5.1) are enzymes that catalyze the hydrolysis of nitriles to the corresponding carbon acids. Among the four known nitrilases of Arabidopsis thaliana, the isoform NIT4 is the most divergent one, and homologs of NITI are also known from species not belonging to the Brassicaceae like Nicotiana tabacum and Oryza sativa. We expressed A. thaliana NIT4 as hexahistidine tag fusion protein in Escherichia coli. The purified enzyme showed a strong substrate specificity for P-cyano-L-alanine (Ala(CN)), an intermediate product of cyanide detoxification in higher plants. Interestingly, not only aspartic acid but also asparagine were identified as products of NIT4-catalyzed Ala(CN) hydrolysis, Asn itself was no substrate for NIT4, indicating that it is not an intermediate but one of two reaction products. NIT4 therefore has both nitrilase and nitrile hydratase activity. Several lines of evidence indicate that the catalytic center for both reactions is the same. The NIT4 homologs of N. tabacum were found to catalyze the same reactions and protein extracts of A. thaliana, N, tabacum and Lupinus angustifolius also converted Ala(CN) to Asp and Asn in vitro. NIT4 may play a role in cyanide detoxification during ethylene biosynthesis because extracts from senescent leaves of A. thaliana showed higher Ala(CN) hydratase/nitrilase activities than extracts from nonsenescent tissue.