Identification of the enantiomeric nature of 2-keto-3-deoxy-galactonate in the catabolic pathway of 3,6-anhydro-L-galactose

Abstract

A novel metabolic pathway of 3,6-anhydro-L-galactose (L-AHG), the main sugar component in red macroalgae, was first discovered in the marine bacterium Vibrio sp. EJY3. L-AHG is converted to 2-keto-3-deoxy-galactonate (KDGal) in two metabolic steps. Here, we identified the enantiomeric nature of KDGal in the L-AHG catabolic pathway via stereospecific enzymatic reactions accompanying the biosynthesis of enantiopure L-KDGal and D-KDGal. Enantiopure L-KDGal and D-KDGal were synthesized by enzymatic reactions derived from the fungal galacturonate and bacterial oxidative galactose pathways, respectively. KDGal, which is involved in the L-AHG pathway, was also prepared. The results obtained from the reactions with an L-KDGal aldolase, specifically acting on L-KDGal, showed that KDGal in the L-AHG pathway exists in an L-enantiomeric form. Notably, we demonstrated the utilization of L-KDGal by Escherichia coli for the first time. E. coli cannot utilize L-KDGal as the sole carbon source. However, when a mixture of L-KDGal and D-galacturonate was used, E. coli utilized both. Our study suggests a stereoselective method to determine the absolute configuration of a compound. In addition, our results can be used to explore the novel L-KDGal catabolic pathway in E. coli and to construct an engineered microbial platform that assimilates L-AHG or L-KDGal as substrates. KEY POINTS: • Stereospecific enzyme reactions were used to identify enantiomeric nature of KDGal • KDGal in the L-AHG catabolic pathway exists in an L-enantiomeric form • E. coli can utilize L-KDGal as a carbon source when supplied with D-galacturonate.

Keywords: 2-Keto-3-deoxy-galactonate; 3,6-Anhydro-L-galactose; Enantiomer; Escherichia coli; Red macroalgae; Stereospecificity.