Glycogen is the primary storage polysaccharide in bacteria and animals. It is a glucose polymer linked by α-1,4 glucose linkages and branched via α-1,6-linkages, with the latter reaction catalyzed by branching enzymes. Both the length and dispensation of these branches are critical in defining the structure, density, and relative bioavailability of the storage polysaccharide. Key to this is the specificity of branching enzymes because they define branch length. Herein, we report the crystal structure of the maltooctaose-bound branching enzyme from the enterobacteria E. coli. The structure identifies three new malto-oligosaccharide binding sites and confirms oligosaccharide binding in seven others, bringing the total number of oligosaccharide binding sites to twelve. In addition, the structure shows distinctly different binding in previously identified site I, with a substantially longer glucan chain ordered in the binding site. Using the donor oligosaccharide chain-bound Cyanothece branching enzyme structure as a guide, binding site I was identified as the likely binding surface for the extended donor chains that the E. coli branching enzyme is known to transfer. Furthermore, the structure suggests that analogous loops in branching enzymes from a diversity of organisms are responsible for branch chain length specificity. Together, these results suggest a possible mechanism for transfer chain specificity involving some of these surface binding sites.

The β-hydroxycarboxylic acid functionality of 3-hydroxypropionic acid (3-HP) makes it a versatile building block chemical that can be converted to industrially relevant chemicals such as acrylic acid. Optimization of existing routes commonly utilizing glucose or glycerol as feedstocks is the most commonly targeted area of 3-HP research. In this study, 3-HP biosynthesis is demonstrated using a unique and novel carbon feedstock, acetylenecarboxylic acid (ACA), that can be C1-derived. ACA may be derived from methane and carbon dioxide by methane dehydrodimerization and subsequent monocarboxylation. A novel variant of tautomerase enzyme Cg10062 is used for transformation of ACA to malonate semialdehyde (MSA), a 3-HP precursor. Cg10062 activity is coupled with NADPH-dependent dehydrogenase YdfG to afford successful synthesis of 3-HP with cofactor regeneration. The two-step three-enzyme synthesis was demonstrated using as low as 0.001 equivalents of NADPH relative to ACA. A similar system was also demonstrated using soluble NADH-dependent MmsB.