来自 Azospirillum baldaniorum 的 PHB 聚合酶(PhbC)的生物信息学和功能分析

IF 3.5 Q3 Biochemistry, Genetics and Molecular Biology
Doris del Carmen Fuentes , Lucía Soto-Urzua , Lino Javier Martínez-Soto , Luis Javier Martínez-Morales
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引用次数: 0

摘要

背景Azospirillum baldaniorum Sp245 能产生聚-β-羟基丁酸酯,这是一种可生物降解的聚合物,其特性类似于聚丙烯等合成热塑性塑料。在合成途径中,聚-β-羟基丁酸合成酶以 3-羟基丁酰-CoA 的硫代酯为底物,并催化其与 HS-CoA 释放的聚合作用。选择其中一个基因进行扩增并克隆到 pEXP5- CT/TOPO® 载体中,然后通过限制性模式、聚合酶链反应和测序对其进行分析。SDS-PAGE 分析确定了 Azospirillum baldaniorum 的 PhbC1 蛋白(AbPhbC1)的分子量。使用抗聚组氨酸单克隆抗体进行 Western 印迹,证实了该蛋白的存在。利用埃尔曼法测定了巯基的浓度,从而确定了 AbPhbC1 粗提取物中的酶活性。还进行了紫外可见光检测。结果硅学分析表明,AbPhbC1 和来自 Azospirillum baldaniorum 的 PhbC2 蛋白(AbPhbC2)保留了聚-β-羟丁酸聚合酶和 α/β 水解酶结构域。在所有四个聚β-羟基烷酸合成酶的中心亚域中,Cys-His-Asp催化三元组高度保守,结构上与已报道的结晶蛋白相似。AbPhbC1 的二聚化亚域是封闭的;AbPhbC2 的二聚化亚域是开放的;AbPhbC2 与 III 类和 IV 类多-β-羟基丙二酸合成酶一样缺少 EC 区。在体外,AbPhbC1 的分子量为 68 kDa。AbPhbC1 对 PHB 的聚合作用是通过定量测定 SH 释放出的 HS-CoA 来检测的。UV-Vis 扫描显示在 264 纳米波长处有一个特征峰。结论硅学分析表明,AbPhbC1 和 AbPhbC2 在结构上是功能性的,只是 AbPhbC2 的活性可能需要 PhaR 亚基;这强烈表明它可能是第四类聚-β-羟基丙酸合成酶。紫外-可见光扫描和核磁共振光谱显示,A. baldaniorum 的 AbPhbC1 酶合成了聚-β-羟基丁酸,表明该酶具有功能。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Bioinformatic and functional analysis of a PHB polymerase (PhbC) from Azospirillum baldaniorum

Background

Azospirillum baldaniorum Sp245 produces poly-β-hydroxybutyrate, a biodegradable polymer with characteristics similar to synthetic thermoplastics, including polypropylene. In the synthesis pathway, the poly-β-hydroxybutyrate synthase enzyme uses thioesters of 3-hydroxy butyryl-CoA as a substrate and catalyzes their polymerization with HS-CoA release.

Methods

A study was conducted using in silico analysis of the two phbC genes of A. baldaniorum Sp245. One was selected for amplification and cloning into the pEXP5- CT/TOPO® vector, which was analysed by restriction pattern, polymerase chain reaction, and sequencing. SDS-PAGE analysis determined the molecular weight of the PhbC1 protein from Azospirillum baldaniorum (AbPhbC1). The presence of the protein was confirmed by Western blotting using anti-polyhistidine monoclonal antibodies. The enzymatic activity in the crude extract of AbPhbC1 was determined by measuring the concentration of sulfhydryl groups using the Ellman method. A UV–Vis assay was performed. To confirm the presence of the poly-β-hydroxybutyrate product, an NMR assay was performed.

Results

In silico analyses, it is revealed that AbPhbC1 and the PhbC2 protein from Azospirillum baldaniorum (AbPhbC2) retain the poly-β-hydroxybutyrate polymerase and α/β hydrolase domain. The Cys-His-Asp catalytic triad is highly conserved in all four polyβ-hydroxyalkanoate synthases in the central subdomain, structurally similar to the reported crystallized proteins. The dimerization subdomain is different; in AbPhbC1, it is in the closed form; in AbPhbC2, it is in the open form; and in AbPhbC2, it lacks the EC region as class III and IV poly-β-hydroxyalcanoate synthases. In vitro, the molecular weight of AbPhbC1 was 68 kDa. The polymerization of PHB by AbPhbC1 was detected by the release of HS-CoA from the quantification of SH. The UV–Vis scan showed a characteristic peak at 264 nm. A comparison of the NMR spectra of the bacterial and commercial poly-β-hydroxybutyrate samples suggested their presence.

Conclusion

In silico analyses suggested that AbPhbC1 and AbPhbC2 are structurally functional, except that AbPhbC2 might require the PhaR subunit for its activity; this strongly suggests that it could be a class IV poly-β-hydroxyalcanoate synthase. UV–Vis scanning and NMR spectroscopy revealed the synthesis of poly-β-hydroxybutyrate by the A. baldaniorum enzyme AbPhbC1, indicating that the enzyme is functional.

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来源期刊
Journal of Genetic Engineering and Biotechnology
Journal of Genetic Engineering and Biotechnology Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
5.70
自引率
5.70%
发文量
159
审稿时长
16 weeks
期刊介绍: Journal of genetic engineering and biotechnology is devoted to rapid publication of full-length research papers that leads to significant contribution in advancing knowledge in genetic engineering and biotechnology and provide novel perspectives in this research area. JGEB includes all major themes related to genetic engineering and recombinant DNA. The area of interest of JGEB includes but not restricted to: •Plant genetics •Animal genetics •Bacterial enzymes •Agricultural Biotechnology, •Biochemistry, •Biophysics, •Bioinformatics, •Environmental Biotechnology, •Industrial Biotechnology, •Microbial biotechnology, •Medical Biotechnology, •Bioenergy, Biosafety, •Biosecurity, •Bioethics, •GMOS, •Genomic, •Proteomic JGEB accepts
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