Molecular Studies to Investigate 4-hydroxy-2-oxoglutarate Metabolism Defects of Primary Hyperoxaluria Type 3

Abstract

Primary hyperoxaluria (PH) is marked by recurrent calcium oxalate kidney stones that form due to the overproduction of oxalate in the liver. PH is caused by mutations affecting enzymes involved in hydroxyproline and glyoxylate metabolism. Of the three PH types, the third type, PH3, is the most recently classified and least thoroughly investigated. PH3 results when mutations prevent 4-hydroxy-2-oxoglutarate aldolase (HOGA) from cleaving 4-hydroxy-2-oxoglutarate (HOG) into pyruvate and glyoxylate. It is proposed that the consequences of PH3 could be lessened by preventing conditions that favor oxalate production downstream of deficient HOGA and by recovering activity of mutant HOGA directly by chemical chaperone treatments. Glyoxylate reductase (GR) is an enzyme with a role in glyoxylate metabolism downstream of HOGA and is inhibited by HOG. Attempts to crystallize GR in complex with HOG were insufficient to produce crystals that diffracted consistently beyond 8 Å. Additionally, a new HPLC assay was developed for monitoring HOGA activity in crude lysates. Results from this assay indicated that exposure to the chemical chaperones TMAO, glycerol, and DMSO during expression in E. coli did not increase the activity of several HOGA mutants, though treatment with 10% glycerol may have increased activity of the R70P mutant to ~80% of WT. Future studies should investigate alternative crystallization methods and additional chaperone treatments.