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

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.

Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites

“…Various approaches have been taken for the synthesis of phosphorylated monosaccharide sugar acids, such as the use of microbial whole cells, selective enzymatic phosphorylation of the corresponding monosaccharide sugar acid, or selective water elimination from phosphorylated monosaccharide sugar acids. This is exemplified with 2-keto-3-deoxy-6-phospho-D-gluconate, abbreviated KDPG, which was prepared from Alcaligenes eutrophus strain H16 F34 lacking KDPG-aldolase activity [ 127 ], from 2-keto-3-deoxy-D-gluconate by kinase-catalyzed phosphorylation [ 128 , 129 ], and through 6-phosphogluconate dehydratase-catalyzed water elimination from 6-phospho-D-gluconate [ 130 ].…”

Selective Biocatalytic Defunctionalization of Raw Materials

“…The synthesis of 2‐keto‐3‐deoxy‐6‐phospho‐ d ‐gluconate has been achieved by the elimination of water from 6‐phospho‐ d ‐gluconate catalyzed by 6‐phosphogluconate dehydratase [71,72] . A variety of dehydratases has enabled also the straightforward synthetic access to various 2‐keto‐3‐deoxy‐aldonic acids with a backbone of six carbon atoms, such as 2‐keto‐3‐deoxy‐ d ‐gluconate and 2‐keto‐3‐deoxy‐ l ‐gluconate, [73–75] or 2‐keto‐3‐deoxy‐ d ‐galactonate and 2‐keto‐3‐deoxy‐ l ‐galactonate [75,76] .…”

“…The synthesis of 2-keto-3-deoxy-6-phospho-d-gluconate has been achieved by the elimination of water from 6-phospho-dgluconate catalyzed by 6-phosphogluconate dehydratase. [71,72] A variety of dehydratases has enabled also the straightforward synthetic access to various 2-keto-3-deoxy-aldonic acids with a backbone of six carbon atoms, such as 2-keto-3-deoxy-dgluconate and 2-keto-3-deoxy-l-gluconate, [73][74][75] or 2-keto-3deoxy-d-galactonate and 2-keto-3-deoxy-l-galactonate. [75,76] Other sugar acid dehydratases have been used for synthesizing 2-keto-3-deoxy-aldonic acids with a backbone of five carbon atoms, such as 2-keto-3-deoxy-d-xylonate, [76,77] 2-keto-3-deoxyl-fuconate, [78] 2-keto-3-deoxy-d-fuconate, and 2-keto-3-deoxy-lrhamnonate, [70] 2-keto-3-deoxy-l-arabinonate, [79] and 2-keto-3deoxy-d-arabinonate.…”

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis

“…The analysis of chemical and biological routes for the synthesis of metabolites often shows large differences in the number of reaction steps required to construct highly and differentially functionalized small molecules. Designing highly selective and straightforward biocatalytic one-step synthesis methods instead of lengthy and challenging chemical routes has been established as the preferred method for preparative access to a large number of valuable metabolites (Richter et al, 2009;Schell et al, 2009;Matsumi et al, 2014;Schoenenberger et al, 2017a;Hardt et al, 2017;Schoenenberger et al, 2018;Krevet et al, 2020;Schoenenberger et al, 2020;Sun et al, 2021). As the finding and selection of routes is a key task in target-oriented synthesis the development of appropriate methodologies has attracted much interest in both chemistry and biology.…”