Structural Analysis and Substrate Specificity of D-Carbamoylase from Pseudomonas.

IF 2.7 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
BioTech Pub Date : 2024-10-03 DOI:10.3390/biotech13040040
Marina Paronyan, Haykanush Koloyan, Hovsep Aganyants, Artur Hambardzumyan, Tigran Soghomonyan, Sona Avetisyan, Sergey Kocharov, Henry Panosyan, Vehary Sakanyan, Anichka Hovsepyan
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Abstract

The synthesis of enantiomeric forms of D-amino acids can be achieved by a two-step "hydantoinase process" based on the sequential catalysis of substrates by specific enzymes, D-carbamoylase and D-hydantoinase. Here, we describe the structural features of D-carbamoylase from Pseudomonas, the encoded gene of which was chemically synthesized and cloned into Escherichia coli. A significant fraction of the overexpressed recombinant protein forms insoluble inclusion bodies, which are partially converted to a soluble state upon treatment with N-lauroylsarcosine or upon incubation of cells at 28 °C. Purified His-tagged protein exhibits the highest activity towards N-carbamoyl-D-alanine and N-carbamoyl-D-tryptophan. Comprehensive virtual analysis of the interactions of bulky carbamylated amino acids with D-carbamoylase provided valuable information. Molecular docking analysis revealed the location of the substrate binding site in the three-dimensional structure of D-carbamoylase. Molecular dynamics simulations showed that the binding pocket of the enzyme in complex with N-carbamoyl-D-tryptophan was stabilized within 100 nanoseconds. The free energy data showed that Arg176 and Asn173 formed hydrogen bonds between the enzyme and substrates. The studies of D-carbamoylases and the properties of our previously obtained D-hydantoinase suggest the possibility of developing a harmonized biotechnological process for the production of new drugs and peptide hormones.

假单胞菌 D-氨基甲酰酶的结构分析和底物特异性
D- 氨基酸对映体的合成可通过两步 "海因酶过程 "实现,该过程基于特定酶(D-氨基甲酰酶和 D-海因酶)对底物的顺序催化。在这里,我们描述了假单胞菌 D-氨基甲酰酶的结构特征,其编码基因是通过化学合成并克隆到大肠杆菌中的。大量过表达的重组蛋白形成不溶性包涵体,经 N-月桂酰肌氨酸处理或细胞在 28 °C下孵育后,包涵体部分转化为可溶状态。纯化的 His 标记蛋白对 N-氨基甲酰-D-丙氨酸和 N-氨基甲酰-D-色氨酸的活性最高。对大体积氨甲酰化氨基酸与 D-氨基甲酰酶的相互作用进行的全面虚拟分析提供了有价值的信息。分子对接分析揭示了 D-氨基甲酰酶三维结构中底物结合位点的位置。分子动力学模拟显示,该酶与 N-氨基甲酰基-D-色氨酸的结合口袋在 100 纳秒内稳定下来。自由能数据显示,Arg176 和 Asn173 在酶和底物之间形成氢键。对 D-氨基甲酰酶的研究以及我们先前获得的 D-海因糖酶的特性表明,有可能开发出一种用于生产新药物和肽类激素的协调生物技术工艺。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
BioTech
BioTech Immunology and Microbiology-Applied Microbiology and Biotechnology
CiteScore
3.70
自引率
0.00%
发文量
51
审稿时长
11 weeks
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