Abstract:
Human dihydrodipicolinate synthase-like protein (DHDPSL) is a protein of unknown function. However, mutations in the Dhdpsl gene were recently linked to primary hyperoxaluria type III, a rare endogenous disorder of oxalate production. Consequently, Dhdpsl was hypothesized to encode a putative aldolase, 4-hydroxy-2-ketoglutarate aldolase, which catalyzes the final step of hydroxyproline catabolism. There is also evidence that DHDPSL is implicated in gluconeogenesis, where it was identified as a specific target for an inhibitor. In as yet unpublished research, the DHDPSL crystal structure was recently solved by Dr. Richard Bunker in the School of Biological Sciences, University of Auckland. This research showed that the structure shares the same fold as bacterial aldolases, with conservation of active site catalytic residues and tetrameric quaternary structure. Despite these similarities, the biological function of human DHDPSL is unknown. The aim of this study was provide a functional assignment for this orphan enzyme. In this study, DHDPSL was for the first time characterized as a bifunctional oxaloacetate decarboxylase and a 4-hydroxy-2-ketoglutarate aldolase. The Km for the oxaloacetate decarboxylase activity for untagged and fusion DHDPSL was 22.27 and 17.25 μM oxaloacetate, respectively. DHDPSL was identified as a 4-hydroxy-2-ketoglutrate aldolase using Fourier Transform Mass Spectrometry (FTMS). The stability, optimal temperature and pH of the oxaloacetate decarboxylase activity was characterised. Further kinetic investigations revealed that the enzyme was inhibited by the TCA metabolites malate and succinate. Divalent cations were also found to be inhibitory, with observed enzyme activity ranging from 50% to complete inhibition. Overall, these results suggest physiologically significant regulatory effects of DHDPSL in the mitochondria. The oxaloacetate decarboxylase activity of DHDPSL could establish a futile cycle with the first step of gluconeogenesis, whereby pyruvate is converted to oxaloacetate. Presumably, DHDPSL must be endogenously regulated such that pyruvate cycling is coordinately controlled. Inhibition of the oxaloacetate decarboxylase pathway by TCA metabolites suggests that physiologically significant mechanisms exist that regulates this pathway. Inhibition of DHDPSL by metal cations also opens up another level of regulation in the mitochondria.