Regulation of human 4-hydroxy-2-oxoglutarate aldolase by pyruvate and α-ketoglutarate: implications for primary hyperoxaluria type-3.

4-hydroxy-2-oxoglutarate aldolase (HOGA1) is a mitochondrial enzyme that plays a gatekeeper role in hydroxyproline metabolism. Its loss of function in humans causes primary hyperoxaluria type 3 (PH3), a rare condition characterised by excessive production of oxalate. In this study, we investigated the significance of the associated oxaloacetate decarboxylase activity which is also catalysed by HOGA1. Kinetic studies using the recombinant human enzyme (hHOGA1) and active site mutants showed both these dual activities utilise the same catalytic machinery with micromolar substrate affinities suggesting that both are operative in vivo. Biophysical and structural studies showed that pyruvate was a competitive inhibitor with an inhibition constant in the micromolar range. By comparison α-ketoglutarate was a weak inhibitor with an inhibition constant in the millimolar range and could only be isolated as an adduct with the active site Lys196 in the presence of sodium borohydride. These studies suggest that pyruvate inhibits HOGA1 activity during gluconeogenesis. We also propose that loss of HOGA1 function could increase oxalate production in PH3 by decreasing pyruvate availability and metabolic flux through the Krebs cycle.

Cellular degradation of 4-hydroxy-2-oxoglutarate aldolase leads to absolute deficiency in primary hyperoxaluria type 3.

Primary hyperoxaluria type-3 is characterized by increased oxalate production caused by mutations in the HOGA1 gene encoding 4-hydroxy-2-oxoglutarate aldolase (HOGA1). How the most commonly occurring mutations affect the cellular fates of the expressed HOGA1 mutants is still unknown. We show that two prevalent recombinant HOGA1 mutants are thermally unstable with evidence for chaperone-mediated degradation when expressed in E. coli. In stably transformed HEK-293 cells, protein expression of the Glu315 deletion mutant only becomes detectable during incubation with a 26S proteasome inhibitor. These findings suggest that failure of chaperone-assisted folding leads to targeted cellular degradation and an absolute absence of HOGA1 function.

Use of a novel microtitration protocol to obtain diffraction-quality crystals of 4-hydroxy-2-oxoglutarate aldolase from Bos taurus.

The enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA) catalyses the retro-aldol degradation of 4-hydroxy-2-oxoglutarate to pyruvate and glyoxylate as part of the hydroxyproline catabolic pathway in mammals. Mutations in the coding region of the human HOGA gene are associated with primary hyperoxaluria type 3, a disease characterized by excessive oxalate production and ultimately stone deposition. Native HOGA was purified from bovine kidney using an improved and streamlined purification protocol from which two crystal forms were obtained using two different approaches. Vapour diffusion using PEG 3350 as a precipitant produced monoclinic crystals that belonged to space group C2 and diffracted to 3.5 Šresolution. By comparison, orthorhombic crystals belonging to space group I222 or I212121 and diffracting to beyond 2.25 Šresolution were obtained using a novel microtitration protocol with ammonium sulfate. The latter crystal form displayed superior diffraction quality and was suitable for structural determination by X-ray crystallography.