Penicillium camemberti galacturonate reductase: C-1 xidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids

Authors: Wagschal, Kurt; JORDAN, DOUGLAS - Retired ARS Employee; Hart-Cooper, William; Chan, Victor

Submitted to: International Journal of Biological Macromolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2019
Publication Date: 11/19/2019
Citation: Wagschal, K.C., Jordan, D.B., Hart-Cooper, W.M., Chan, V.J. 2019. Penicillium camemberti galacturonate reductase: C-1 xidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids. International Journal of Biological Macromolecules. 153:1090-1098. https://doi.org/10.1016/j.ijbiomac.2019.10.239.
DOI: https://doi.org/10.1016/j.ijbiomac.2019.10.239

Interpretive Summary: The world-wide yearly production of orange juice ~1.7 x 106 metric tons/year generates significant food processing waste that can be a disposal problem, which could be lessened by generating new products from this under-utilized waste stream. Dried citrus peel contains ~27% D-galacturonic acid (GalUA), the main sugar acid component of pectin Likewise, the pulp waste steam from sugar beet refining, with worldwide production estimated to be ~ 250 x 106 metric tons, contains ~24% pectin and thus large amounts of GalUA. Enzymes can be an environmentally benign, cost effective method to biosynthetically convert GalUA in 3 steps to ascorbic acid (vitamin C), and we identified an enzyme that catalyzes the first step towards biosynthetic vitamin C, to a molecule termed galactonic acid. In order to use the enzyme in processes for vitamin C biosynthetic production, it is necessary to measure its rate of conversion of GalUA in the presence of both GalUA and its product. We discovered that the product of the enzyme unexpectedly allows the enzyme to continue working at a high rate in the presence of large concentrations of GalUA, which would otherwise normally slow down the reaction. These results are important for designing enzyme pathways for vitamin C biosynthesis.

Technical Abstract: The enzyme galacturonate oxidoreductase PcGOR from Penicillium camemberti reduces glucuronic and galacturonic acids to their corresponding aldonic acids, important reactions in pectin catabolism and ascorbate biosynthesis. The enzyme was characterized and found to be active on both glucuronic acid and galacturonic acid, with similar specificities (kcat/Km) using the preferred cofactor NADPH. Substrate inhibition was observed for galacturonate, whereas glucuronate showed only minor substrate inhibition, and the product and its lactone from reaction with glucuronate, L-gulonate and L-gulono-1,4-lactone, were found to be competitive inhibitors with Ki in the low mM range. The product and its lactone from reaction with galacturonate, L-galactonate and L-galactono-1,4-lactone, were likewise inhibitors but surprisingly also appeared to allosterically mitigate substrate inhibition, with the lactone having a greater effect than the acid, and furthermore since the next enzyme en route to ascorbate forms L-galactono-1,4-lactone from L-galactonate, its activity could possibly become entrained with that of PcGOR at high substrate and product loadings.