HYDROXYPROLINE DEHYDROGENASE: A PROMISING TARGET FOR TREATING ALL THREE FORMS OF PRIMARY HYPEROXALURIA
The Primary Hyperoxalurias (PH) are rare disorders of glyoxylate metabolism that cause the formation of calcium oxalate kidney stones. Currently, treatments for PH are limited. Available treatments focus on easing the symptoms of PH, not treating the disease itself. Hydroxyproline metabolism is a potential pathway for treating for PH because it makes a significant contribution to the pool of glyoxylate formed in the body. It has been hypothesized that hydroxyproline dehydrogenase (HYPDH), traditionally referred to as hydroxyproline oxidase and proline dehydrogenase 2, is an ideal target for treating PH because HYPDH is a unique and initial step in the hydroxyproline metabolism pathway. Also, it appears that the inhibition of HYPDH is harmless. This thesis is the first report of the expression, purification, and biochemical properties of human HYPDH. A brute force screening approach for the purification of constructs allowed for the determination of the catalytic core of HYPDH. This catalytic core was tested. It was found that HYPDH prefers hydroxyproline over any other substrate and that HYPDH binds hydroxyproline very tightly. A sodium sulfite titration and kinetic studies with quinone electron acceptors demonstrated that HYPDH is a poor oxidase, thus making the enzyme a dehydrogenase. Mutagenesis studies of the active site determined that Ala186, Gln234, and Ser485 are residues crucial for substrate binding. Initially, commercially available proline analogs were screened for inhibitory activity. Three inhibitors were identified, and each had an IC50 values between 1.5 mM-3 mM. Further optimization of the analogs revealed several more inhibitors of increased potency, with IC50 values in the low micromolar range. In an effort to determine the structure of HYPDH, many variants of HYPDH, such as various N-terminal truncations and different species of HYPDH, were extensively screened under several conditions. No crystals were obtained. It was hypothesized that HYPDH requires the presence of P5CDH and part of its N-terminus in order to crystallize. Thus, a crucial future direction is to determine the crystal structure of HYPDH. It is hoped that this body of work will help in the design of inhibitors of HYPDH to treat PH.
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