Departamento de Bioquímica y Biología Molecular, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, Córdoba, Spain.
Departamento de Botánica, Ecología y Fisiología Vegetal, Edificio Celestino Mutis, Campus de Rabanales, Universidad de Córdoba, Córdoba, Spain.
Microbiol Spectr. 2021 Dec 22;9(3):e0077721. doi: 10.1128/Spectrum.00777-21. Epub 2021 Nov 3.
3-Cyanoalanine and cyanohydrins are intermediate nitriles produced in cyanide degradation pathways in plants and bacteria. 3-Cyanoalanine is generated from cyanide by the 3-cyanoalanine synthase, an enzyme mainly characterized in cyanogenic plants. NIT4-type nitrilases use 3-cyanoalanine as a substrate, forming ammonium and aspartate. In some organisms, this enzyme also generates asparagine through an additional nitrile hydratase activity. The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 assimilates cyanide through an intermediate cyanohydrin, which is further converted into ammonium by the nitrilase NitC. This bacterium also contains three additional nitrilases, including Nit4. In this work, a proteomic analysis of CECT5344 cells grown with 3-cyanoalanine as the sole nitrogen source has revealed the overproduction of different proteins involved in nitrogen metabolism, including the nitrilase NitC. In contrast, the nitrilase Nit4 was not induced by 3-cyanoalanine, and it was only overproduced in cells grown with a cyanide-containing jewelry-manufacturing residue. Phenotypes of single and double mutant strains defective in or/and revealed the implication of the nitrilase NitC in the assimilation of 3-cyanoalanine and suggest that the 3-cyanoalanine assimilation pathway in CECT5344 depends on the presence or absence of cyanide. When cyanide is present, 3-cyanoalanine is assimilated via Nit4, but in the absence of cyanide, a novel pathway for 3-cyanoalanine assimilation, in which the nitrilase NitC uses the nitrile generated after deamination of the α-amino group from 3-cyanoalanine, is proposed. Nitriles are organic cyanides with important industrial applications, but they are also found in nature. 3-Cyanoalanine is synthesized by plants and some bacteria to detoxify cyanide from endogenous or exogenous sources, but this nitrile may be also involved in other processes such as stress tolerance, nitrogen and sulfur metabolism, and signaling. The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344 grows with 3-cyanoalanine as the sole nitrogen source, but it does not use this nitrile as an intermediate in the cyanide assimilation pathway. In this work, a quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to study, for the first time, the response to 3-cyanoalanine at the proteomic level. Proteomic data, together with phenotypes of different nitrilase-defective mutants of CECT5344, provide evidence that in the absence of cyanide, the nitrilase Nit4 is not involved in 3-cyanoalanine assimilation, and instead, the nitrilase NitC participates in a novel alternative 3-cyanoalanine assimilation pathway.
3-氰基丙氨酸和氰醇是植物和细菌中氰化物降解途径产生的中间腈。3-氰基丙氨酸由 3-氰基丙氨酸合酶从氰化物生成，该酶主要存在于含氰植物中。NIT4 型腈酶以 3-氰基丙氨酸为底物，形成铵和天冬氨酸。在一些生物体中，该酶还通过额外的腈水解酶活性生成天冬酰胺。耐碱假单胞菌 Pseudomonas pseudoalcaligenes CECT5344 通过一种中间氰醇同化氰化物，该氰醇进一步被腈酶 NitC 转化为铵。该细菌还含有另外三种腈酶，包括 Nit4。在这项工作中，对仅以 3-氰基丙氨酸为唯一氮源生长的 CECT5344 细胞进行的蛋白质组学分析表明，涉及氮代谢的不同蛋白质过度表达，包括 NitC 腈酶。相比之下，3-氰基丙氨酸没有诱导 Nit4 的产生，只有在含有含氰珠宝制造残余物的细胞中才会过度产生。或/和 缺失的单突变和双突变菌株的表型表明，NitC 腈酶参与了 3-氰基丙氨酸的同化，并且表明 3-氰基丙氨酸在 CECT5344 中的同化途径取决于氰化物的存在与否。当氰化物存在时，3-氰基丙氨酸通过 Nit4 同化，但在没有氰化物的情况下，提出了一种用于 3-氰基丙氨酸同化的新途径，其中 NitC 腈酶使用 3-氰基丙氨酸的α-氨基脱氨后生成的腈。腈是具有重要工业应用的有机氰化物，但它们也存在于自然界中。3-氰基丙氨酸由植物和一些细菌合成，用于从内源性或外源性来源解毒氰化物，但这种腈可能也参与其他过程，如应激耐受、氮和硫代谢以及信号转导。氰化物降解菌 Pseudomonas pseudoalcaligenes CECT5344 以 3-氰基丙氨酸为唯一氮源生长，但它不将此腈用作氰化物同化途径中的中间产物。在这项工作中，通过液相色谱-串联质谱 (LC-MS/MS) 进行了定量蛋白质组学分析，首次在蛋白质组学水平上研究了 3-氰基丙氨酸的反应。蛋白质组学数据以及 CECT5344 的不同 Nit4 缺陷突变体的表型表明，在没有氰化物的情况下，Nit4 腈酶不参与 3-氰基丙氨酸同化，而是 NitC 腈酶参与新的替代 3-氰基丙氨酸同化途径。
