title: Proline dehydrogenase 2 (PRODH2) is a hydroxyproline dehydrogenase (HYPDH) and molecular target for treating primary hyperoxaluria.
abstract: The primary hyperoxalurias (PH), types 1-3, are disorders of glyoxylate metabolism that result in increased oxalate production and calcium oxalate stone formation. The breakdown of trans-4-hydroxy-L-proline (Hyp) from endogenous and dietary sources of collagen makes a significant contribution to the cellular glyoxylate pool. Proline dehydrogenase 2 (PRODH2), historically known as hydroxyproline oxidase, is the first step in the hydroxyproline catabolic pathway and represents a drug target to reduce the glyoxylate and oxalate burden of PH patients. This study is the first report of the expression, purification, and biochemical characterization of human PRODH2. Evaluation of a panel of N-terminal and C-terminal truncation variants indicated that residues 157-515 contain the catalytic core with one FAD molecule. The 12-fold higher k(cat)/K(m) value of 0.93 M⁻¹·s⁻¹ for Hyp over Pro demonstrates the preference for Hyp as substrate. Moreover, an anaerobic titration determined a K(d) value of 125 μM for Hyp, a value ~1600-fold lower than the K(m) value. A survey of ubiquinone analogues revealed that menadione, duroquinone, and CoQ₁ reacted more efficiently than oxygen as the terminal electron acceptor during catalysis. Taken together, these data and the slow reactivity with sodium sulfite support that PRODH2 functions as a dehydrogenase and most likely utilizes CoQ₁₀ as the terminal electron acceptor in vivo. Thus, we propose that the name of PRODH2 be changed to hydroxyproline dehydrogenase (HYPDH). Three Hyp analogues were also identified to inhibit the activity of HYPDH, representing the first steps toward the development of a novel approach to treat all forms of PH.
authors.affiliation: *Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27101, U.S.A.
authors.forename: Candice B
authors.initials: CB
authors.lastname: Summitt
authors.affiliation: *Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27101, U.S.A.
authors.forename: Lynnette C
authors.initials: LC
authors.lastname: Johnson
authors.affiliation: *Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27101, U.S.A.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Jönsson
authors.affiliation: *Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27101, U.S.A.
authors.forename: Derek
authors.initials: D
authors.lastname: Parsonage
authors.affiliation: †Department of Urology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.
authors.forename: Ross P
authors.initials: RP
authors.lastname: Holmes
authors.affiliation: *Center for Structural Biology and Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC 27101, U.S.A.
authors.forename: W Todd
authors.initials: WT
authors.lastname: Lowther
mesh_terms: D055162:Biocatalysis; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005663:Furans / Q000494:pharmacology / Q000627:therapeutic use; D006801:Humans; D006909:Hydroxyproline / Q000737:chemistry / Q000378:metabolism*; D006960:Hyperoxaluria, Primary / Q000188:drug therapy / Q000201:enzymology*; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011394:Proline Oxidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D009626:Terminology as Topic; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D005420:Flavoproteins; D005663:Furans; D008024:Ligands; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014451:Ubiquinone; D005182:Flavin-Adenine Dinucleotide; C063698:tetrahydro-2-furancarboxylic acid; D011392:Proline; D011394:Proline Oxidase; C452185:proline dehydrogenase (oxidase) 2; C024989:coenzyme Q10; D006909:Hydroxyproline