Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

Sabine Krevet; Lu Shen; Timon Bohnen; Bernhard Schoenenberger; Roland Meier; Markus Obkircher; Klara Bangert; Rudolf Koehling; Eric Allenspach; Roland Wohlgemuth; Bettina Siebers; Christopher Bräsen

doi:10.3389/fbioe.2020.00185

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus

Front Bioeng Biotechnol. 2020 Mar 20:8:185. doi: 10.3389/fbioe.2020.00185. eCollection 2020.

Affiliations

¹ Molecular Enzyme Technology and Biochemistry, Environmental Microbiology and Biotechnology (EMB), Centre for Water and Environmental Research, Faculty of Chemistry, University of Duisburg-Essen, Essen, Germany.
² Member of Merck Group, Sigma-Aldrich Production GmbH, Buchs, Switzerland.
³ Institute of Molecular and Industrial Biotechnology, Technical University Lodz, Lodz, Poland

Abstract

The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a V_max of 61.6 U mg^-1 and a K_M of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

Keywords: 2-keto-3-deoxy-6-phosphogluconate; 6-phosphogluconate; 6-phosphogluconate dehydratase; Caulobacter crescentus; Entner-Doudoroff pathway; biocatalytic dehydration; metabolite.