Journal: The Journal of Biological Chemistry

Article Title: Structural and functional consequences of aspartate/asparagine-β-hydroxylase variants causing Traboulsi syndrome

doi: 10.1016/j.jbc.2025.111008

Figure Lengend Snippet: Locations of Traboulsi syndrome–associated AspH variants . A , the AspH catalyzed reaction; ( B ) view from a reported wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structure (PDB ID: 6YYW ) showing positions of Traboulsi syndrome–associated active site variations (R735W/R735Q and R688Q) and tetratricopeptide repeat (TPR) domain variations (G434V) in yellow . Colors: gray : AspH 315–758 ; teal : carbon backbone of 2OG; violet : Mn, substitutes for the active cofactor Fe(II); slate blue : carbon backbone of the hFX-derived hFX-EGFD1 86–124 -4S peptide ( C ). AspH, aspartate/asparagine-β-hydroxylase; EGFD, epidermal growth factor–like domain; hFX, human coagulation factor X; 2OG, 2-oxoglutarate; PDB, Protein Data Bank.

Article Snippet: To obtain crystal seeds of wt AspH, a mixture of wt AspH 315–758 (374 μM), Mn(II) (1 mM), 2OG (2 mM), and hFX-EGFD1 86–124 -4S (0.8 mM) in buffer (50 mM Hepes, pH 7.5, 150 mM NaCl) was incubated at 4 °C for 15 min prior to centrifugation using a MicroCL 21R centrifuge (Thermo Fisher Scientific; 18,800 g , 4 °C, 5 min).

Techniques: Derivative Assay, Coagulation

Journal: The Journal of Biological Chemistry

Article Title: Structural and functional consequences of aspartate/asparagine-β-hydroxylase variants causing Traboulsi syndrome

doi: 10.1016/j.jbc.2025.111008

Figure Lengend Snippet: Activities of Traboulsi syndrome–associated active site AspH variants are substantially reduced compared with wt AspH . A , using wt AspH ( orange circles ; 0.1 μM) or G434V AspH (0.1 μM), hFX-EGFD1 86–124 -4S (4.0 μM), l -ascorbic acid (LAA; 100 μM), 2OG (20 μM), Fe(II) (20 μM) in buffer (25 mM Hepes, 50 mM NaCl, pH 7.5); ( B ) using wt AspH ( orange circles ; 0.1 μM) or an AspH variant (R688Q, black triangles ; R735Q, brown inverse triangles ; and R735W, red hexagons ; 0.1 μM), hFX-EGFD1 86–124 -4S (4.0 μM), LAA (100 μM), 2OG (20 μM), Fe(II) (20 μM) in buffer (25 mM Hepes, 50 mM NaCl, pH 7.5); ( C ) using wt AspH ( orange circles ; 0.2 μM) or an AspH variant (G434V, green diamonds ; R688Q, black triangles ; R735Q, brown inverse triangles ; and R735W, red hexagons ; 0.2 μM), hFX-EGFD1 86–124 -4S (4.0 μM), LAA (400 μM), 2OG (30 μM or 2000 μM for R735Q and R735W AspH), Fe(II) (100 μM) in buffer (25 mM Mes, pH 6.0); and blue boxes : no-enzyme control. Measurement times were normalized to the first sample injection analyzed after the addition of AspH to the substrate mixture ( t = 0 min), by which time low levels of hydroxylation were manifest; results are means of three independent runs (n = 3; mean ± SD). AspH, aspartate/asparagine-β-hydroxylase; EGFD, epidermal growth factor–like domain; hFX, human coagulation factor X; 2OG, 2-oxoglutarate.

Article Snippet: To obtain crystal seeds of wt AspH, a mixture of wt AspH 315–758 (374 μM), Mn(II) (1 mM), 2OG (2 mM), and hFX-EGFD1 86–124 -4S (0.8 mM) in buffer (50 mM Hepes, pH 7.5, 150 mM NaCl) was incubated at 4 °C for 15 min prior to centrifugation using a MicroCL 21R centrifuge (Thermo Fisher Scientific; 18,800 g , 4 °C, 5 min).

Techniques: Variant Assay, Control, Injection, Coagulation

Journal: The Journal of Biological Chemistry

Article Title: Structural and functional consequences of aspartate/asparagine-β-hydroxylase variants causing Traboulsi syndrome

doi: 10.1016/j.jbc.2025.111008

Figure Lengend Snippet: Kinetic parameters for the AspH variant–catalyzed hydroxylation of hFX-EGFD1 86–124 -4S using SPE–MS . A – I , determination of the G434V ( green diamonds ), R688Q ( black triangles ), and R735Q ( brown inverse triangles ) AspH variant v max app and K m app values for ( A – C ) LAA, ( D – F ) 2OG, and ( G – I ) Fe(II). Assays employed 0.1 μM (G434V) or 0.2 μM (R688Q, R735Q) AspH variant; details are described in the section. Catalysis by the tested AspH variants did not depend on LAA. The initial hydroxylation rates used to determine kinetic parameters are shown in . The results are means of three independent runs (n = 3; mean ± SD). AspH, aspartate/asparagine-β-hydroxylase; EGFD, epidermal growth factor–like domain; hFX, human coagulation factor X; LAA, l -ascorbic acid; 2OG, 2-oxoglutarate; SPE–MS, solid phase extraction coupled to mass spectrometry.

Article Snippet: To obtain crystal seeds of wt AspH, a mixture of wt AspH 315–758 (374 μM), Mn(II) (1 mM), 2OG (2 mM), and hFX-EGFD1 86–124 -4S (0.8 mM) in buffer (50 mM Hepes, pH 7.5, 150 mM NaCl) was incubated at 4 °C for 15 min prior to centrifugation using a MicroCL 21R centrifuge (Thermo Fisher Scientific; 18,800 g , 4 °C, 5 min).

Techniques: Variant Assay, Coagulation, Extraction, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Structural and functional consequences of aspartate/asparagine-β-hydroxylase variants causing Traboulsi syndrome

doi: 10.1016/j.jbc.2025.111008

Figure Lengend Snippet: The conformation of 2OG in complex with the Traboulsi syndrome–associated R735Q and R735W AspH variants differs from that observed in complex with wt AspH . Colors: gray : wt and variant AspH 315–758 ; teal : carbon backbone of 2OG; lavender : Mn; yellow : carbon backbone of the hFX-EGFD1 86–124 -4S peptide ( C ); red : oxygen; blue : nitrogen; and w: water. A – C , representative Polder omit density maps contoured to 3.0σ around ( A ) 2OG and Mn in the wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structure, ( B ) 2OG, Mn, and Q735 in the R735Q AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structure, and ( C ) 2OG, Mn, and W735 in the R735W AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structure; ( D – F ) superimposition of active site views of ( D ) the R735Q AspH:2OG:Mn:hFX-EGFD1 86–124 -4S (colors: gray : R735Q AspH; teal : 2OG; lavender : Mn; yellow : carbon backbone of hFX-EGFD1 86–124 -4S) and wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structures (colors: brown : R735Q AspH; orange : 2OG; pink : Mn; light green : carbon backbone of hFX-EGFD1 86–124 -4S); ( E ) the R735W AspH:2OG:Mn:hFX-EGFD1 86–124 -4S (colors: ochre : R735Q AspH; magenta : 2OG; lavender : Mn; light blue : carbon backbone of hFX-EGFD1 86–124 -4S) and wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structures, and ( F ) the R735Q AspH:2OG:Mn:hFX-EGFD1 86–124 -4S and R735W AspH:2OG:Mn:hFX-EGFD1 86–124 -4S structures. AspH, aspartate/asparagine-β-hydroxylase; EGFD, epidermal growth factor–like domain; hFX, human coagulation factor X; 2OG, 2-oxoglutarate.

Article Snippet: To obtain crystal seeds of wt AspH, a mixture of wt AspH 315–758 (374 μM), Mn(II) (1 mM), 2OG (2 mM), and hFX-EGFD1 86–124 -4S (0.8 mM) in buffer (50 mM Hepes, pH 7.5, 150 mM NaCl) was incubated at 4 °C for 15 min prior to centrifugation using a MicroCL 21R centrifuge (Thermo Fisher Scientific; 18,800 g , 4 °C, 5 min).

Techniques: Variant Assay, Coagulation

Journal: The Journal of Biological Chemistry

Article Title: Structural and functional consequences of aspartate/asparagine-β-hydroxylase variants causing Traboulsi syndrome

doi: 10.1016/j.jbc.2025.111008

Figure Lengend Snippet: 3-Methyl-2OG and 2OS bind to the Traboulsi syndrome–associated R688Q and R735Q AspH variants in a similar manner as 2OG binds to wt AspH . Colors: gray : R688Q and R735Q AspH 315–758 ; lavender : Mn; yellow : carbon backbone of the hFX-EGFD1 86–124 -4S peptide ( C ); red : oxygen; blue : nitrogen; and w: water. A , representative Polder omit electron density maps contoured to 3.0σ around (3 R )-3-methyl-2OG ( 1 ; salmon ), Mn ( lavender ), and Q688 in the R688Q AspH: 1 :Mn:hFX-EGFD1 86–124 -4S structure; ( B and C ) superimposition of active site views of the R688Q AspH: 1 :Mn:hFX-EGFD1 86–124 -4S structure with ( B ) the reported wt AspH: 1 :Mn:hFX-EGFD1 86–124 -4S (colors: light pink : wt AspH; olive : ( R )- 1 ; purple : Mn; blue : carbon backbone of hFX-EGFD1 86–124 -4S; PDB ID: 6YYX ) and ( C ) the improved wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S (colors: brown : wt AspH; orange : 2OG; pink : Mn; light green : carbon backbone of hFX-EGFD1 86–124 -4S) structures. D , representative Polder omit electron density maps contoured to 3.0σ around 2-oxosuberate (2OS; 12 ; lemon green ), Mn ( lavender ), and Q735 in the R735Q AspH:2OS:Mn:hFX-EGFD1 86–124 -4S structure; ( E and F ) superimposition of active site views of the R735Q AspH:2OS:Mn:hFX-EGFD1 86–124 -4S structure with ( E ) the R735Q AspH:2OG:Mn:hFX-EGFD1 86–124 -4S (colors: light pink : R735Q AspH; teal : 2OG; purple : Mn; and blue : carbon backbone of hFX-EGFD1 86–124 -4S) and ( F ) the improved wt AspH:2OG:Mn:hFX-EGFD1 86–124 -4S (colors: brown : wt AspH; orange : 2OG; pink : Mn; light green : carbon backbone of hFX-EGFD1 86–124 -4S) structures. AspH, aspartate/asparagine-β-hydroxylase; EGFD, epidermal growth factor–like domain; hFX, human coagulation factor X; 2OG, 2-oxoglutarate; 2OS, 2-oxosuberate.

Article Snippet: To obtain crystal seeds of wt AspH, a mixture of wt AspH 315–758 (374 μM), Mn(II) (1 mM), 2OG (2 mM), and hFX-EGFD1 86–124 -4S (0.8 mM) in buffer (50 mM Hepes, pH 7.5, 150 mM NaCl) was incubated at 4 °C for 15 min prior to centrifugation using a MicroCL 21R centrifuge (Thermo Fisher Scientific; 18,800 g , 4 °C, 5 min).

Techniques: Coagulation