Identification and characterization of 3,6-anhydro-L-galactose dehydrogenases belonging to the aldehyde dehydrogenase superfamily from marine and soil microorganisms

Abstract

The complete hydrolysis of agarose produces its monomeric sugars, D-galactose and 3,6-anhydro-Lgalactose (L-AnG). Although enzymes of D-galactose metabolism are well characterized, those involved in L-AnG metabolism have not yet been investigated. In this study, we report the identification and characterization of L-AnG dehydrogenase (L-AnGDH), an aldehyde dehydrogenase (ALDH), catalyzing the first step of L-AnG degradation. To compare substrate and cofactor specificities of L-AnGDH, two L-AnGDH genes obtained from the marine bacterium Postechiella marina (Pm_L-AnGDH) and the soil bacterium Streptomyces coelicolor (Sc_L-AnGDH) were cloned and expressed in E. coli. Whereas the recombinant Pm_L-AnGDH and Sc_L-AnGDH were similar in their oligomeric state (homotetramer) and optimum reaction conditions (30°C, pH 8.0), the two enzymes were distinguishable by their substrate and cofactor specificities. Sc_L-AnGDH catalyzed the oxidation of L-AnG using both NAD + and NADP + , with a preference for NAD + . It also catalyzed the dehydrogenation of L-glyceraldehyde, glycolaldehyde, and L-lactaldehyde in the presence of NAD + . On the other hand, Pm_L-AnGDH showed exclusive selectivity towards NADP + and did not oxidize aldehydes other than L-AnG and L-glyceraldehyde. The phylogenetic analysis of amino sequences indicated that L-AnGDH belongs to a novel subfamily within the ALDH superfamily. To our knowledge, this is the first report on the characterization of L-AnGDH.

Unusual metabolism of 3,6-anhydro-L-galactose in Vibrio sp. EJY3 and in E. coli containing two Vibrio sp. EJY3 genes

“…In summary, bioinformatics analysis and experimental data on L-AnGDH and L-AnGACI from P. marina M091 [1][2][3] clearly showed that the reaction products of LAnGDH and L-AnGACI are L-form metabolites (Fig. 1A).…”

“…Recently, the 3,6-anhydro-L-galactose dehydrogenase (LAnGDH) [2] and 3,6-anhydro-L-galactonate cycloisomerase (L-AnGACI) [3] from Postechiella marina M091 have been characterized. P. marina M091, which was isolated from a seawater sample obtained at Damupo beach in Pohang, Korea, is a novel agar-degrading microorganism belonging to a new genus within the family Flavobacteriaceae [9].…”

“…L-AnGDH is an aldehyde dehydrogenase catalyzing the first step of the L-AnG pathway and oxidizes L-AnG to 3,6-anhydro-L-galactonate (L-AnGA) in the presence of NAD(P) + [1,2]. L-AnG has an open-chain aldehyde structure that resembles α-hydroxyaldehydes such as glyceraldehyde and lactaldehyde [1] and hence the reaction product of L-AnGDH must be a carboxylate derivative of L-AnG, i.e.…”

“…Lee et al recently used comparative genomic analysis of three agar-degrading microorganisms, which are Postechiella marina M091, Pseudoalteromonas atlantica T6c, and Streptomyces coelicolor A3 (2), to identify the metabolic pathway of L-AnG [1]. They combined bioinformatic analysis and experimental data to reveal that L-AnG is metabolized to pyruvate and D-glyceraldehyde-3-phosphate via six enzyme-catalyzed reactions (Fig.…”

“…1A). They also have conducted in-depth studies on 3,6-anhydro-L-galactose dehydrogenase (LAnGDH) [2] and 3,6-anhydro-L-galactonate cycloisomerase (L-AnGACI) [3], which are the first and the second step of the L-AnG pathway of three agar-degrading microorganisms. The metabolic pathway of L-AnG in another marine microorganism, Vibrio sp.…”