Laboratório Nacional de Computação Científica, Petrópolis, Rio de Janeiro, Brazil.
Centro Multiusuário de Inovação Biomolecular, Departamento de Física, Universidade, Estadual Paulista (UNESP), São José do Rio Preto, 15054-000, SP, Brazil.
J Mol Graph Model. 2019 Jan;86:35-42. doi: 10.1016/j.jmgm.2018.10.005. Epub 2018 Oct 9.
In this work we performed several in silico analyses to describe the relevant structural aspects of an enzyme N-Carbamoyl-d-amino acid amidohydrolase (d-NCAase) encoded on the genome of the Brazilian strain CPAC 15 (=SEMIA 5079) of Bradyrhizobium japonicum, a nonpathogenic species belonging to the order Rhizobiales. d-NCAase has wide applications particularly in the pharmaceutical industry, since it catalyzes the production of d-amino acids such as D-p-hydroxyphenylglycine (D-HPG), an intermediate in the synthesis of β-lactam antibiotics. We applied a homology modelling approach and 50 ns of molecular dynamics simulations to predict the structure and the intersubunit interactions of this novel d-NCAase. Also, in order to evaluate the substrate binding site, the model was subjected to 50 ns of molecular dynamics simulations in the presence of N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) (a d-NCAase canonical substrate) and water-protein/water-substrate interactions analyses were performed. Overall, the structural analysis and the molecular dynamics simulations suggest that d-NCAase of B. japonicum CPAC-15 has a homodimeric structure in solution. Here, we also examined the substrate specificity of the catalytic site of our model and the interactions with water molecules into the active binding site were comprehensively discussed. Also, these simulations showed that the amino acids Lys123, His125, Pro127, Cys172, Asp174 and Arg176 are responsible for recognition of ligand in the active binding site through several chemical associations, such as hydrogen bonds and hydrophobic interactions. Our results show a favourable environment for a reaction of hydrolysis that transforms N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) into the active compound D-p-hydroxyphenylglycine (D-HPG). This work envisage the use of d-NCAase from the Brazilian Bradyrhizobium japonicum strain CPAC-15 (=SEMIA 5079) for the industrial production of D-HPG, an important intermediate for semi-synthesis of β-lactam antibiotics such as penicillins, cephalosporins and amoxicillin.
在这项工作中，我们进行了几项计算机分析，以描述编码于巴西布拉德氏菌 CPAC 15（=SEMIA 5079）基因组中的酶 N-碳酰基-d-氨基酸酰胺水解酶（d-NCAase）的相关结构方面。d-NCAase 在制药工业中有广泛的应用，因为它可以催化 d-氨基酸的生产，如 D-p-羟基苯甘氨酸（D-HPG），这是β-内酰胺抗生素合成的中间体。我们应用同源建模方法和 50 ns 的分子动力学模拟来预测这种新型 d-NCAase 的结构和亚基间相互作用。此外，为了评估底物结合位点，我们将模型在 N-碳酰基-d-p-羟基苯甘氨酸（Cp-HPG）（d-NCAase 的典型底物）存在下进行了 50 ns 的分子动力学模拟，并对水-蛋白/水-底物相互作用进行了分析。总体而言，结构分析和分子动力学模拟表明，来自巴西根瘤菌 CPAC-15 的 d-NCAase 在溶液中具有同源二聚体结构。在这里，我们还检查了我们模型的催化位点的底物特异性，并全面讨论了与活性结合位点中的水分子的相互作用。此外，这些模拟表明，氨基酸 Lys123、His125、Pro127、Cys172、Asp174 和 Arg176 通过氢键和疏水相互作用等几种化学结合，负责识别活性结合位点中的配体。我们的结果显示出有利于水解反应的环境，将 N-碳酰基-d-p-羟基苯甘氨酸（Cp-HPG）转化为活性化合物 D-p-羟基苯甘氨酸（D-HPG）。这项工作设想使用来自巴西布拉德氏菌 CPAC-15（=SEMIA 5079）的 d-NCAase 用于 D-HPG 的工业生产，D-HPG 是半合成β-内酰胺抗生素如青霉素、头孢菌素和阿莫西林的重要中间体。
