3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli’s cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.

Previously, we have identified and characterized 4,6-α-glucanotransferase enzymes of the glycosyl hydrolase (GH) family 70 (GH70) that cleave (α1→4)-linkages in amylose and introduce (α1→6)-linkages in linear chains. The 4,6-α-glucanotransferase of Lactobacillus reuteri 121, for instance, converts amylose into an isomalto/malto-polysaccharide (IMMP) with 90% (α1→6)-linkages. Over the years, also, branching sucrase enzymes belonging to GH70 have been characterized. These enzymes use sucrose as a donor substrate to glucosylate dextran as an acceptor substrate, introducing single -(1→2,6)-α-d-Glcp-(1→6)- (Leuconostoc citreum enzyme) or -(1→3,6)-α-d-Glcp-(1→6)-branches (Leuconostoc citreum, Leuconostoc fallax, Lactobacillus kunkeei enzymes). In this work, we observed that the catalytic domain 2 of the L. kunkeei branching sucrase used not only dextran but also IMMP as the acceptor substrate, introducing -(1→3,6)-α-d-Glcp-(1→6)-branches. The products obtained have been structurally characterized in detail, revealing the addition of single (α1→3)-linked glucose units to IMMP (resulting in a comb-like structure). The in vitro digestibility of the various α-glucans was estimated with the glucose generation rate (GGR) assay that uses rat intestinal acetone powder to simulate the digestive enzymes in the upper intestine. Raw wheat starch is known to be a slowly digestible carbohydrate in mammals and was used as a benchmark control. Compared to raw wheat starch, IMMP and dextran showed reduced digestibility, with partially digestible and indigestible portions. Interestingly, the digestibility of the branching sucrase modified IMMP and dextran products considerably decreased with increasing percentages of (α1→3)-linkages present. The treatment of amylose with 4,6-α-glucanotransferase and branching sucrase/sucrose thus allowed for the synthesis of amylose/starch derived α-glucans with markedly reduced digestibility. These starch derived α-glucans may find applications in the food industry.

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