Yamanashi Tomoya, Kim Seung-Young, Hara Hirofumi, Funa Nobutaka
a Graduate Division of Nutritional and Environmental Sciences , University of Shizuoka , Shizuoka , Japan.
Biosci Biotechnol Biochem. 2015;79(5):830-5. doi: 10.1080/09168451.2014.993915. Epub 2015 Jan 3.
The β-ketoadipate pathway is a major pathway involved in the catabolism of the aromatic compounds in microbes. The recent progress in genome sequencing has led to a rapid accumulation of genes from the β-ketoadipate pathway in the available genetic database, yet the functions of these genes remain uncharacterized. In this study, the protocatechuate branch of the β-ketoadipate pathway of Rhodococcus jostii was reconstituted in vitro. Analysis of the reaction products of PcaHG, PcaB, and PcaL was achieved by high-performance liquid chromatography. These reaction products, β-ketoadipate enol-lactone, 3-carboxy-cis,cis-muconate, γ-carboxymuconolactone, muconolactone, and β-ketoadipate, were further characterized using LC-MS and nuclear magnetic resonance. In addition, the in vitro reaction of PcaL, a bidomain protein consisting of γ-carboxy-muconolactone decarboxylase and β-ketoadipate enol-lactone hydrolase activities, was demonstrated for the first time. This work provides a basis for analyzing the catalytic properties of enzymes involved in the growing number of β-ketoadipate pathways deposited in the genetic database.
β-酮己二酸途径是微生物中参与芳香族化合物分解代谢的主要途径。基因组测序的最新进展导致β-酮己二酸途径中的基因在现有遗传数据库中迅速积累，但这些基因的功能仍未得到表征。在本研究中，体外重建了约氏红球菌β-酮己二酸途径的原儿茶酸分支。通过高效液相色谱法分析了PcaHG、PcaB和PcaL的反应产物。使用液相色谱-质谱联用仪和核磁共振进一步表征了这些反应产物，即β-酮己二酸烯醇内酯、3-羧基-顺,顺-粘康酸、γ-羧基粘康内酯、粘康内酯和β-酮己二酸。此外，首次证明了具有γ-羧基粘康内酯脱羧酶和β-酮己二酸烯醇内酯水解酶活性的双结构域蛋白PcaL的体外反应。这项工作为分析遗传数据库中越来越多的β-酮己二酸途径中所涉及酶的催化特性提供了基础。
