Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, India.
Department of Biosciences and Bioengineering, Indian Institute of Technology-Bombay, Powai, Mumbai, India
Appl Environ Microbiol. 2018 Jan 2;84(2). doi: 10.1128/AEM.02115-17. Print 2018 Jan 15.
sp. strains C5pp and C7 degrade carbaryl as the sole carbon source. Carbaryl hydrolase (CH) catalyzes the hydrolysis of carbaryl to 1-naphthol and methylamine. Bioinformatic analysis of , encoding CH, in C5pp predicted it to have a transmembrane domain (Tmd) and a signal peptide (Sp). In these isolates, the activity of CH was found to be 4- to 6-fold higher in the periplasm than in the cytoplasm. The recombinant CH (rCH) showed 4-fold-higher activity in the periplasm of The deletion of Tmd showed activity in the cytoplasmic fraction, while deletion of both Tmd and Sp (Tmd+Sp) resulted in expression of the inactive protein. Confocal microscopic analysis of expressing a (Tmd+Sp)-green fluorescent protein (GFP) fusion protein revealed the localization of GFP into the periplasm. Altogether, these results indicate that Tmd probably helps in anchoring of polypeptide to the inner membrane, while Sp assists folding and release of CH in the periplasm. The N-terminal sequence of the mature periplasmic CH confirms the absence of the Tmd+Sp region and confirms the signal peptidase cleavage site as Ala-Leu-Ala. CH purified from strains C5pp, C7, and rCHΔ(Tmd)a were found to be monomeric with molecular mass of ∼68 to 76 kDa and to catalyze hydrolysis of the ester bond with an apparent and in the range of 98 to 111 μM and 69 to 73 μmol · min · mg, respectively. The presence of low-affinity CH in the periplasm and 1-naphthol-metabolizing enzymes in the cytoplasm of spp. suggests the compartmentalization of the metabolic pathway as a strategy for efficient degradation of carbaryl at higher concentrations without cellular toxicity of 1-naphthol. Proteins in the periplasmic space of bacteria play an important role in various cellular processes, such as solute transport, nutrient binding, antibiotic resistance, substrate hydrolysis, and detoxification of xenobiotics. Carbaryl is one of the most widely used carbamate pesticides. Carbaryl hydrolase (CH), the first enzyme of the degradation pathway which converts carbaryl to 1-naphthol, was found to be localized in the periplasm of spp. Predicted transmembrane domain and signal peptide sequences of were found to be functional in and to translocate CH and GFP into the periplasm. The localization of low-affinity CH into the periplasm indicates controlled formation of toxic and recalcitrant 1-naphthol, thus minimizing its accumulation and interaction with various cellular components and thereby reducing the cellular toxicity. This study highlights the significance of compartmentalization of metabolic pathway enzymes for efficient removal of toxic compounds.
菌株 C5pp 和 C7 可将carbaryl 作为唯一碳源进行降解。Carbaryl 水解酶 (CH) 可催化 carbaryl 水解生成 1-萘酚和甲胺。对 C5pp 中编码 CH 的基因进行生物信息学分析，预测其具有跨膜结构域 (Tmd) 和信号肽 (Sp)。在这些分离物中，CH 的活性在周质中比细胞质中高 4-6 倍。重组 CH (rCH) 在周质中的活性提高了 4 倍。Tmd 的缺失导致细胞质部分的活性，而 Tmd 和 Sp（Tmd+Sp）的缺失则导致无活性蛋白的表达。表达 (Tmd+Sp)-绿色荧光蛋白 (GFP) 融合蛋白的分析表明 GFP 定位于周质。总之，这些结果表明 Tmd 可能有助于将多肽锚定在内膜上，而 Sp 则有助于 CH 在周质中的折叠和释放。成熟周质 CH 的 N 端序列证实了 Tmd+Sp 区域的缺失，并证实信号肽切割位点为 Ala-Leu-Ala。从菌株 C5pp、C7 和 rCHΔ(Tmd)a 中纯化得到的 CH 均为单体，分子量约为 68 至 76 kDa，并催化酯键水解，表观 和 在 98 至 111 μM 和 69 至 73 μmol·min·mg 范围内，分别。 spp. 周质中存在低亲和力 CH 和细胞质中 1-萘酚代谢酶，表明代谢途径的区室化是一种在没有 1-萘酚细胞毒性的情况下高效降解 carbaryl 的策略，因为 carbaryl 浓度较高。细菌周质中的蛋白质在各种细胞过程中发挥着重要作用，例如溶质运输、营养结合、抗生素抗性、底物水解和外源性化合物的解毒。Carbaryl 是最广泛使用的氨基甲酸酯类农药之一。Carbaryl 水解酶 (CH) 是降解途径的第一个酶，可将 carbaryl 转化为 1-萘酚，发现其定位于 spp. 的周质中。预测的跨膜结构域和信号肽序列在 和 GFP 转运到周质中被发现具有功能。低亲和力 CH 的定位到周质表明对有毒和难降解的 1-萘酚进行了控制形成，从而最大限度地减少其积累和与各种细胞成分的相互作用，从而降低细胞毒性。本研究强调了代谢途径酶的区室化对于有效去除有毒化合物的重要性。
