Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases

BMC Biotechnology

Research Article

Risa Hemmi¹  Kenji Miyamoto¹  Rena Momma¹  Volker Sieber²  Barbara Beer²  Jochen Schmid²  André Pick² 

[1] Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, 2238522, Yokohama, Japan;Technical University of Munich, Straubing Center of Science, Chair of Chemistry of Biogenic Resources, Schulgasse 16, 94315, Straubing, Germany;

关键词: Keto-deoxy-D-Glucarate;    Acinetobacter baylyi;    Comamonas testosteroni;    Polaromonas naphthalenivorans;    Dehydratase;   

DOI  :  10.1186/s12896-016-0308-3

 received in 2016-05-29, accepted in 2016-10-21,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundHexuronic acids such as D-galacturonic acid and D-glucuronic acid can be utilized via different pathways within the metabolism of microorganisms. One representative, the oxidative pathway, generates α-keto-glutarate as the direct link entering towards the citric acid cycle. The penultimate enzyme, keto-deoxy glucarate dehydratase/decarboxylase, catalyses the dehydration and decarboxylation of keto-deoxy glucarate to α-keto-glutarate semialdehyde. This enzymatic reaction can be tracked continuously by applying a pH-shift assay.ResultsTwo new keto-deoxy glucarate dehydratases/decarboxylases (EC 4.2.1.41) from Comamonas testosteroni KF-1 and Polaromonas naphthalenivorans CJ2 were identified and expressed in an active form using Escherichia coli ArcticExpress(DE3). Subsequent characterization concerning Km, kcat and thermal stability was conducted in comparison with the known keto-deoxy glucarate dehydratase/decarboxylase from Acinetobacter baylyi ADP1. The kinetic constants determined for A. baylyi were Km 1.0 mM, kcat 4.5 s−1, for C. testosteroni Km 1.1 mM, kcat 3.1 s−1, and for P. naphthalenivorans Km 1.1 mM, kcat 1.7 s−1. The two new enzymes had a slightly lower catalytic activity (increased Km and a decreased kcat) but showed a higher thermal stability than that of A. baylyi. The developed pH-shift assay, using potassium phosphate and bromothymol blue as the pH indicator, enables a direct measurement. The use of crude extracts did not interfere with the assay and was tested for wild-type landscapes for all three enzymes.ConclusionsBy establishing a pH-shift assay, an easy measurement method for keto-deoxy glucarate dehydratase/decarboxylase could be developed. It can be used for measurements of the purified enzymes or using crude extracts. Therefore, it is especially suitable as the method of choice within an engineering approach for further optimization of these enzymes.

【 授权许可】

CC BY   
© The Author(s). 2016