{"title":"Burkholderia sp. AJ110349和Variovorax sp. AJ110348 n -乙酰基-(R)-β-苯丙氨酸酰化酶的表达、纯化、结晶及结构测定","authors":"Yuki Kato, Hisashi Kawasaki, Tsuyoshi Nakamatsu, Namio Matsuda, Ryo Natsume","doi":"10.1107/S2053230X23000730","DOIUrl":null,"url":null,"abstract":"<p><i>N</i>-Acetyl-(<i>R</i>)-β-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of <i>N</i>-acetyl-(<i>R</i>)-β-phenylalanine to produce enantiopure (<i>R</i>)-β-phenylalanine. In previous studies, <i>Burkholderia</i> sp. AJ110349 and <i>Variovorax</i> sp. AJ110348 were isolated as (<i>R</i>)-enantiomer-specific <i>N</i>-acetyl-(<i>R</i>)-β-phenylalanine acylase-producing organisms and the properties of the native enzyme from <i>Burkholderia</i> sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure–function relationships of the enzymes derived from both organisms. The recombinant <i>N</i>-acetyl-(<i>R</i>)-β-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the <i>Burkholderia</i> enzyme belonged to space group <i>P</i>4<sub>1</sub>2<sub>1</sub>2, with unit-cell parameters <i>a</i> = <i>b</i> = 112.70–112.97, <i>c</i> = 341.50–343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of <i>N</i>,<i>N</i>-dimethylformamidase from <i>Paracoccus</i> sp. strain DMF. The crystals of the <i>Variovorax</i> enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the <i>N</i>-acetyl-(<i>R</i>)-β-phenylalanine acylases were clarified to be dimeric in solution.</p>","PeriodicalId":7029,"journal":{"name":"Acta crystallographica. Section F, Structural biology communications","volume":"79 3","pages":"70-78"},"PeriodicalIF":1.1000,"publicationDate":"2023-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1107/S2053230X23000730","citationCount":"0","resultStr":"{\"title\":\"Expression, purification and crystallization of N-acetyl-(R)-β-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. 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The recombinant <i>N</i>-acetyl-(<i>R</i>)-β-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the <i>Burkholderia</i> enzyme belonged to space group <i>P</i>4<sub>1</sub>2<sub>1</sub>2, with unit-cell parameters <i>a</i> = <i>b</i> = 112.70–112.97, <i>c</i> = 341.50–343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of <i>N</i>,<i>N</i>-dimethylformamidase from <i>Paracoccus</i> sp. strain DMF. The crystals of the <i>Variovorax</i> enzyme grew as twinned crystals and were not suitable for structure determination. 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引用次数: 0
摘要
n -乙酰基-(R)-β-苯丙氨酸酰化酶是一种水解n -乙酰基-(R)-β-苯丙氨酸酰胺键生成对映纯(R)-β-苯丙氨酸的酶。在前人的研究中,分离到Burkholderia sp. AJ110349和Variovorax sp. AJ110348为(R)-对映体特异性n -乙酰基-(R)-β-苯丙氨酸酰化酶产生菌,并对Burkholderia sp. AJ110349中天然酶的性质进行了表征。在这项研究中,进行了结构分析,以研究这两种生物衍生的酶的结构-功能关系。采用悬挂滴气相扩散法在多种结晶溶液条件下结晶重组n -乙酰基-(R)-β-苯丙氨酸酰化酶。Burkholderia酶的晶体属于空间群P41212,其单位细胞参数a = b = 112.70-112.97, c = 341.50-343.32 Å,在不对称单元中可能含有两个亚基。用Se-SAD方法解析了晶体结构,表明不对称单元中的两个亚基形成了二聚体。每个亚基由3个结构域组成,它们与副球菌菌株DMF的N,N-二甲基甲酰胺酶大亚基的结构域具有相似性。Variovorax酶的晶体生长为双晶,不适合用于结构测定。利用在线静态光散射分析的粒径排除色谱,澄清了n -乙酰-(R)-β-苯丙氨酸酰化酶在溶液中的二聚体。
Expression, purification and crystallization of N-acetyl-(R)-β-phenylalanine acylases derived from Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 and structure determination of the Burkholderia enzyme
N-Acetyl-(R)-β-phenylalanine acylase is an enzyme that hydrolyzes the amide bond of N-acetyl-(R)-β-phenylalanine to produce enantiopure (R)-β-phenylalanine. In previous studies, Burkholderia sp. AJ110349 and Variovorax sp. AJ110348 were isolated as (R)-enantiomer-specific N-acetyl-(R)-β-phenylalanine acylase-producing organisms and the properties of the native enzyme from Burkholderia sp. AJ110349 were characterized. In this study, structural analyses were carried out in order to investigate the structure–function relationships of the enzymes derived from both organisms. The recombinant N-acetyl-(R)-β-phenylalanine acylases were crystallized by the hanging-drop vapor-diffusion method under multiple crystallization solution conditions. The crystals of the Burkholderia enzyme belonged to space group P41212, with unit-cell parameters a = b = 112.70–112.97, c = 341.50–343.32 Å, and were likely to contain two subunits in the asymmetric unit. The crystal structure was solved by the Se-SAD method, suggesting that two subunits in the asymmetric unit form a dimer. Each subunit was composed of three domains, and they showed structural similarity to the corresponding domains of the large subunit of N,N-dimethylformamidase from Paracoccus sp. strain DMF. The crystals of the Variovorax enzyme grew as twinned crystals and were not suitable for structure determination. Using size-exclusion chromatography with online static light-scattering analysis, the N-acetyl-(R)-β-phenylalanine acylases were clarified to be dimeric in solution.
期刊介绍:
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