Sharma Rishabh, Keshari Deepa, Singh Kumar Sachin, Singh Sudheer Kumar
Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow - 226031, India.
Microbiology Division, CSIR-Central Drug Research Institute, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow - 226031, India; Academy of Scientific and Industrial Research (AcSIR), New Delhi, India.
Biochem Biophys Res Commun. 2017 Jun 10;487(4):892-897. doi: 10.1016/j.bbrc.2017.04.149. Epub 2017 May 2.
Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).
氨基酸生物合成已成为新药物靶点的一个来源，因为许多对氨基酸营养缺陷的细菌菌株在体内条件下无法增殖。支链氨基酸（BCAAs）对结核分枝杆菌（Mtb）的存活很重要，其生物合成缺陷的菌株在小鼠体内生长减弱。苏氨酸脱水酶（IlvA）是一种依赖磷酸吡哆醛（PLP）的酶，催化异亮氨酸生物合成的第一步。对结核分枝杆菌H37Ra（Mtb-Ra）中注释为编码IlvA的MRA_1571进行了克隆、表达和纯化。随后将纯化的蛋白用于开发酶活性测定并研究其生化特性。此外，构建了大肠杆菌BL21（DE3）IlvA基因敲除菌株（大肠杆菌-ΔilvA），并用Mtb-Ra ilvA表达构建体（pET32a-ilvA）进行基因互补，以构建互补大肠杆菌菌株（大肠杆菌-ΔilvA + pET32a-ilvA）。大肠杆菌-ΔilvA在基本培养基中生长失败，但在大肠杆菌-ΔilvA + pET32a-ilvA中观察到生长恢复。在异亮氨酸存在的情况下，大肠杆菌-ΔilvA的生长也得到恢复。IlvA定位研究检测到其在细胞壁和膜组分中的分布，在细胞质组分中的含量相对较少。在感染巨噬细胞的野生型（WT）Mtb-Ra中，在72小时时观察到IlvA的最大表达。此外，与WT和互补菌株（KDC）相比，Mtb-Ra IlvA基因敲低（KD）在巨噬细胞中的存活率降低。
