Figure 1.
Metabolism of 4-hydroxyproline and glyoxylate.
Four mitochondrial enzymes are responsible for 4-hydroxproline (4-Hyp) breakdown: hydroxyproline oxidase (HPOX), Δ1-pyrroline-5-carboxylate dehydrogenase (1P5CDH), aspartate aminotransferase (AspAT), and 4-hydroxy-2-oxoglutarate aldolase (HOGA). The terminal HOGA reaction cleaves 4-hydroxy-2-oxoglutarate (HOG) into pyruvate and glyoxylate. Glyoxylate is metabolized either to glycolate by glyoxylate reductase (GR) in the mitochondria and cytoplasm or to glycine by peroxisomal alanine-glyoxylate aminotransferase (AGT). AGT and GR are mutated within primary hyperoxaluria (type 1 and 2, respectively) patients resulting in the buildup of glyoxylate and its conversion by lactate dehydrogenase (LDH) to oxalate, a key component of kidney stones.
Figure 2.
Identification of human HOGA and its sequence relationship to DHDPS enzymes.
(A) SDS-PAGE analysis of bovine HOGA purified from kidney and human HOGA expressed in E. coli. Lane 1, bovine HOGA; lane 2, human HOGA with an N-terminal, 6-His tag; lane 3, protein molecular weight ladder indicated in kDa. (B) Sequence comparison of human HOGA with bovine HOGA (90.2% identity), B. anthracis DHDPS (31.1%), M. tuberculosis DHDPS (26.6%), and E. coli DHDPS (22.3%). Putative catalytic residues for hHOGA (Tyr140, Tyr168, and Lys196) are indicated by green circles. Blue circles indicate additional active site residues that were assessed by site-directed mutagenesis to evaluate their contribution to substrate specificity and catalysis. HOGA mutations identified within PH3 patients are denoted with orange circles [12], [13].
Figure 3.
Comparison of the HOGA, KDPGA, and DHDPS reactions.
The utilization of a Lys residue and Schiff base intermediate with pyruvate for catalysis is shared between the enzymes. However, the preferred reaction directions are opposite, i.e., HOGA and KDPGA perform cleavage reactions, while the DHDPS enzymes utilize a condensation reaction between pyruvate and (S)-aspartate-β-semialdehyde to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinate (HTPA). The wavy bond on the 4-position of HOG indicates that the enzyme can cleave both the S- and R-forms of HOGA [15], [18], [22].
Figure 4.
Characterization of recombinant hHOGA variants.
(A) Representative analytical ultracentrifugation analysis of His-tag free HOGA at one rotor speed (10,500 rpm) and three different concentrations: black triangles, 1.25 mg ml-1; gray squares, 0.82 mg ml−1; open circles, 0.43 mg ml−1. The solid lines were obtained by a fit of these data and those obtained in a replicate experiment to a single ideal species model, which yielded MW = 97,210±810; the residuals are shown above. (B) Circular dichroism analysis of all hHOGA variants within this study. Buffer conditions: 0.2 mg ml−1 HOGA variant, 20 mM HEPES pH 7.5, 100 mM NaCl, 25°C. Data are presented as molar ellipticity vs. wavelength.
Table 1.
Crystallographic data and refinement statistics.
Figure 5.
(A) Homo-tetrameric organization of hHOGA. The tetramer consists of a dimer of dimers (orange and gray). (B) The hHOGA monomer. Two orthogonal views illustrate the overall architecture, TIM barrel fold, and C-terminal helical bundle. Coloring is as follows: gray, α-helices; orange, β-strands; light-orange, loop regions.
Figure 6.
Active site structure of hHOGA.
(A) Pyruvate-Schiff base complex. The 1.97 Å resolution, 2Fo-Fc electron density map is shown in gray and contoured to 1.5 σ. The pyruvate molecule is highlighted by a green Fo-Fc omit map contoured to 6.4 σ. Atom colors are as follows: blue, nitrogen; light-orange, carbon atoms for hHOGA; green, carbon atoms for pyruvate; red, oxygen. (B) Comparison to the unliganded active site. Coloring for pyruvate bound hHOGA is the same as in (A). White coloring is used for unbound hHOGA. Tyr140′ from the adjacent monomer are colored in a darker shade of orange and gray, respectively. A water molecule adjacent to Lys196 is depicted as a red sphere in the unbound hHOGA structure.
Figure 7.
Structural comparisons between hHOGA and E. coli DHDPS.
(A) Superposition of hHOGA and E. coli DHDPS (PDB ID: 3DUO) pyruvate-bound active sites (RMSD = 0.19 Å for Cα atoms) [28]. hHOGA coloring is the same as in Fig. 6. DHDPS carbon atoms are colored in pale cyan and cyan (adjacent subunit). Carbon atoms for pyruvate bound to DHDPS are colored gray. Hydrogen bonds are shown using dashed lines for hHOGA (gray) and DHDPS (blue). Amino acid residues for the two enzymes are indicated in the following order: hHOGA/DHDPS. (B) Model of the HOG•hHOGA complex. HOG carbon atoms and putative hydrogen bonds to hHOGA are colored yellow (R-form) and cyan (S-form).
Table 2.
Kinetic parameters.
Figure 8.
Proposed mechanism for hHOGA catalysis.
The enzyme residues are highlighted in bold. See text for details.
Figure 9.
Location of mutations identified within hHOGA of PH3 patients.
(A) Close-up view of one hHOGA monomer. The cartoon of the protein has been made partially transparent so that the PH3 mutations (spheres; see legend) are highlighted. The G-X-X-G-E motif is illustrated as a blue cartoon tube. The portion of the protein structure leading from the P190L variant to the active site Lys196-pyruvate adduct (stick rendering) is shown in magenta. (B) Three orthogonal views of the hHOGA tetramer illustrate the proximity of the mutations to the monomer-monomer (red dashed line) and dimer-dimer interfaces (blue dashed line). The red and blue squares indicate when an axis is projecting out toward the viewer.