L-Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides
L-Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides
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DOI：
10.1128/aem.01003-13
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发表时间：
2013-08-01
影响因子：
4.4
通讯作者：
Kino, Kuniki
中科院分区：
文献类型：
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作者：
Arai, Toshinobu;Arimura, Yasuhiro;Kino, Kuniki
功能性多肽有望成为改善我们生活质量的有益化合物。为了满足对功能性多肽日益增长的需求，我们利用微生物酶研究了多肽的合成。L-氨基酸连接酶(LAL)以一种依赖于三磷酸腺苷的方式催化无保护氨基酸的缩合，适用于发酵生产。因此，LAL是一种很有希望实现低成本合成的酶。为了获得具有新的底物特异性的LAL，我们重点研究了与双肽类植物毒素TAB的生物合成有关的LAL。从丁香假单胞菌NBRC14081中克隆了Tabs基因，并在大肠杆菌中高效表达。所生产的重组Tabs蛋白显示出任何已知LAL中最广泛的底物特异性；当检测二肽合成时，它检测到231种氨基酸底物组合中的136种。此外，还鉴定了一些新的底物特异性，发现不常见的氨基酸，如L-吡哌酸、羟基-L-脯氨酸和β-丙氨酸是可以接受的底物。此外，动力学分析和短时间内的反应监测表明，TAB在N端和C端显示出明显的底物选择性，这使得有可能特异性地合成没有副产物的多肽，如同源多肽和反序杂多肽。TAB特异性地合成了以下功能肽，包括它们的前体：L-精氨基-L-苯丙氨酸(降压作用，得率，62%)，L-亮氨基-L-异亮氨酸(抗抑郁作用，得率，77%)，L-谷氨酰基-L-色氨酸(具有抗血管生成活性的L-谷氨酰基-L-色氨酸的前体，得率，54%)，L-亮氨基-L-丝氨酸(增强盐度，得率，83%)，以及L-谷氨酰基-L-苏氨酸(增强盐度，得率，96%)。此外，我们的结果还为烟草毒素的生物合成提供了新的见解。
Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and beta-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect; yield, 62%), L-leucyl-L-isoleucine (antidepressive effect; yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity; yield, 54%), L-leucyl-L-serine (enhances saltiness; yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.