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首页> 外文期刊>RSC Advances >Recombinant L-glutaminase obtained from Geobacillus thermodenitrificans DSM-465: characterization and in silico elucidation of conserved structural domains
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Recombinant L-glutaminase obtained from Geobacillus thermodenitrificans DSM-465: characterization and in silico elucidation of conserved structural domains

机译：从Geobacillus Thermodentifificans DSM-465获得的重组L-谷氨酰胺酶：表征和硅释放保守的结构域

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摘要

Glutaminase (GLS) is an enzyme essential for amino acid metabolism; in particular, it acts as a catalyst in glutaminolysis, a reaction exploited by the malignant cells to meet the nutrient requirements for their accelerated growth and proliferation. Via regulating the initial reaction of the glutaminolysis pathway, glutaminase offers an intriguing target for the development of anticancer drugs. In the present study, we produced a recombinant glutaminase from Geobacillus thermodenitrificans DSM-465 in E. coli. The enzyme was purified to electrophoretic homogeneity, with 40% recovery and 22.36 fold purity. It exhibited a molecular weight of 33 kDa, with an optimum pH and temperature of 9 and 70 degrees C, respectively. The K-M value of the purified enzyme was 104 mu M for L-glutamine. A 3D model was built for the enzyme using Swiss-Model and subjected to molecular docking with the substrate and potential inhibitors. Moreover, the subject enzyme was compared with the human kidney type GLS-K by ConSurf and TM-align servers for evolutionary conserved residues and structural domains. Despite having less than 40% amino acid identity, the superimposed monomers of both enzymes exhibited similar to 94% structural identity. With a positional difference, the active site residues Ser65, Asn117, Glu162, Asn169, Tyr193, Tyr245, and Val263 found in the bacterial enzyme were also conserved in the human GLS-K. Molecular docking results have shown that CB-839 is the best inhibitor for GLS-GT and UPGL00004 is the best inhibitor for GLS-K, as designated by the binding free energy changes, i.e. Delta G -388.7 kJ mol(-1) and Delta G -375 kJ mol(-1), respectively. Moreover, six potential inhibitory molecules were ranked according to their binding free energy change values for both enzymes. The information can be used for the in vivo anticancer studies.
机译：谷氨酰胺酶（GLS）是氨基酸代谢必需的酶;特别是，它用作谷氨酸溶解的催化剂，恶性细胞利用的反应，以满足其加速增长和增殖的营养要求。通过调节谷氨酸杆菌途径的初始反应，谷氨酰胺酶为抗癌药物的发育提供了一种有趣的目标。在本研究中，我们在大肠杆菌中制备了来自Geobacillus Thermodentififififififirificans DSM-465的重组谷氨酰胺酶。将酶纯化为电泳均匀性，回收率40％，纯度22.36倍。它分子量为33kDa，分别为最佳pH和温度为9和70℃。纯化酶的K-M值为L-谷氨酰胺为104μm。使用Swiss-Model构建用于酶的3D模型，并与基材和潜在抑制剂进行分子对接。此外，将受试者酶与人肾型GLS-K进行比较，用于进化保守残留物和结构域。尽管氨基酸同一性小于40％，但两种酶的叠加单体表现出类似于94％的结构身份。对于位置差异，在人gls-k中也保守了在细菌酶中发现的活性位点残留物Ser65，Asn117，Glu162，Asn169，Tyr193，Tyr245和Val263。分子对接结果表明，CB-839是GLS-GT的最佳抑制剂，UPG100004是GLS-K的最佳抑制剂，如结合自由能变化，即DELTA G-38.7 kJ摩尔（-1）和DELTA g -375 kj mol（-1）。此外，根据其两种酶的结合可自由能量变化值排序六种潜在的抑制分子。这些信息可用于体内抗癌研究。

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  • 来源
    《RSC Advances》 |2019年第8期|共10页
  • 作者

    Shah Luqman; Nadeem Muhammad Shahid; Khan Jalaluddin Azam; Zeyadi Mustafa A.; Zamzami Mazin A.; Mohammed Kaleemuddin;

  • 作者单位

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

    King Abdulaziz Univ Dept Biochem Fac Sci Bldg A 90 Jeddah 21589 Saudi Arabia;

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