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

Abstract

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 K m, k cat 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 K m 1.0 mM, k cat 4.5 s−1, for C. testosteroni K m 1.1 mM, k cat 3.1 s−1, and for P. naphthalenivorans K m 1.1 mM, k cat 1.7 s−1. The two new enzymes had a slightly lower catalytic activity (increased K m and a decreased k cat) 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0308-3) contains supplementary material, which is available to authorized users.

Alone at last! – Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum

“…In the genome of C. glutamicum , the open reading frame of ksaD overlaps with that of cg0536 encoding for a putative 5-dehydro-4-deoxyglucarate dehydratase. This finding hints towards a potential role of KsaD in a putative oxidative pathway for the utilization of sugar acids such as d -galacturonic acid or d -glucuronic acid as theses pathways require α-ketoglutarate semialdehyde dehydrogenase- and 5-dehydro-4-deoxyglucarate dehydratase activities (Richard and Hilditch, 2009; Pick et al., 2016). It sounds reasonable that C. glutamicum has such a catabolic pathway as these sugar derivatives typically to be found in pectin-rich fruits and vegetables such as grapes, apples, bean sprouts should be readily available in the natural habitat of this soil bacterium (Li et al., 2016).…”

Process Development of Enantioselective Imine Reductase-Catalyzed Syntheses of Pharmaceutically Relevant Pyrrolidines

“…Identification of IREDs Catalyzing the Reduction of 2-Aryl-Substituted Pyrrolines. In order to identify IREDs capable of reducing 2-aryl-substituted pyrrolines, a colorimetric pH-shift assay 25 developed by the Sieber group and adapted by Zumbragel et al 26 was utilized. By means of this assay, the reduction of 2-phenyl-1-pyrroline (1a) in the presence of 31 imine reductases was examined.…”

“…Then, the deep well plates were centrifuged at 4000g for 30 min at 4 °C to obtain the crude extract as a supernatant, which was used in the colorimetric pH-shift assay. The colorimetric shift assay was performed according to a protocol described in ref 25.…”

Enantioselective reduction of sulfur-containing cyclic imines through biocatalysis

“…coli BL21 (DE3) 22 , 29 . As a fast screening methodology a colorimetric pH shift assay 33 developed by the Sieber group was used. This assay is based on a pH shift (visualized by bromthymol blue), which is caused by gluconic acid formed by consumption of the substrate and in situ-cofactor-regeneration (Fig.…”