Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant
Prolyl Aminopeptidase from Streptomyces thermoluteus subsp. fuscus Strain NBRC14270 and Synthesis of Proline-Containing Peptides by Its S144C Variant
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DOI：
10.1128/aem.01242-10
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发表时间：
2010-07
影响因子：
4.4
通讯作者：
Yukihiro Yamamoto;H. Usuki;M. Iwabuchi;T. Hatanaka
中科院分区：
文献类型：
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作者：
Yukihiro Yamamoto;H. Usuki;M. Iwabuchi;T. Hatanaka
摘要我们专门研究了一种胞外肽酶，即脯氨酸氨基肽酶(PAP)，作为合成含有脯氨酸的多肽的靶标。来自热链霉菌亚种的一种PAP通过基于序列的筛选获得Fuscus NBRC14270(PAP14270)。在PAP14270的基础上，用Cys(ScPAP14270)取代了144Ser，获得了氨解活性。与野生型PAP14270不同的是，scPAP14270产生了Pro-Pro与Pro-Pro的聚合物。采用液-质联用(LC/MS)对产物质量进行了确证。考察了pH值、底物浓度、反应时间等因素对反应的影响。此外，还发现底物专一性与在scPAP14270催化的氨解中酰基受体的对数D值之间存在相关性。结果表明，二肽的合成在弱酸性环境中进行，环化和聚合反应在碱性条件下进行。此外，结果表明，除羟脯氨酸苄酯(Hyp-OBzl)外，几乎所有对数值大于0的氨基酸酯都可以被识别为酰基受体。这些发现支持将PAPS用作生产具有生理活性的脯氨酸多肽的工具。
ABSTRACT We specifically examined an exopeptidase, prolyl aminopeptidase (PAP), as a target for synthesis of proline-containing peptides. A PAP from Streptomyces thermoluteus subsp. fuscus NBRC14270 (PAP14270) was obtained using sequence-based screening. From PAP14270, 144Ser was replaced by Cys (scPAP14270) to give aminolysis activity. In contrast to wild-type PAP14270, scPAP14270 produced a polymer of proline benzyl ester and cyclo[Pro-Pro]. The product mass was confirmed using liquid chromatography-mass spectrometry (LC/MS). Several factors affecting the reaction, such as the pH, concentration of the substrate, and reaction time, were measured to determine their effects. Furthermore, a correlation was found between substrate specificity in proline peptide synthesis and the log D value of acyl acceptors in aminolysis catalyzed by scPAP14270. Results showed that dipeptide synthesis proceeded in a weakly acidic environment and that cyclization and polymerization occurred under alkaline conditions. Furthermore, results suggest that almost all amino acid esters whose log D value is greater than 0, except hydroxyproline benzyl ester (Hyp-OBzl), can be recognized as acyl acceptors. These findings support the use of PAPs as a tool for production of physiologically active proline peptides.