Kinetic parameters of human aspartate/asparagine–β-hydroxylase suggest that it has a possible function in oxygen sensing

Abstract

Human aspartate/asparagine–β-hydroxylase (AspH) is a 2-oxoglutarate (2OG)–dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor–like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1–2, 3–4, 5–6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.

Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-β-hydroxylase

“…One possible explanation for its inhibitory effect could be that activated bleomycin A 2 , which is a known oxidant, 42 alters the assay redox equilibrium in the assay mixture, and thus indirectly inhibits AspH, which is known to be sensitive towards subtle changes in the redox environment. 30 Although further work is required, this possibility is in agreement with the results that altered 2OG, substrate peptide or Fe(II) assay concentrations did not affect bleomycin A 2 potency ( Table 2 , entry 3) and that bleomycin A 2 did not seem to bind AspH efficiently (ΔT m = −0.8 ± 0.4 °C; Supporting Fig. S4 ).…”

“… 36 AspH substrate- (hFX-CP 101–119 ; Supporting Fig. S1 b), 2OG-, and Fe(II)-concentrations close to their Michaelis ( K m ) constants 30 were employed and substrate depletion/product formation (+16 Da) was monitored by SPE-MS to identify potent AspH inhibitors ( Supporting Fig. S2 and Supporting Data Sheet).…”

“…2 ). 30 The Abbott-developed Bcl-2 inhibitor ABT-737 55 ( Table 1 , entry 13) was added to the panel due to its structural similarity with navitoclax and venetoclax. The AspH inhibition assays were of good quality which high signal-to-noise (S/N) ratios and high Z'-factors 56 (>0.75 for each plate) manifest ( Supporting Fig.…”

“…To investigate whether the mechanisms by which the identified eleven small-molecule APIs inhibit AspH involve competition with 2OG or substrate for AspH binding or non-specific Fe(II)-chelation, their IC 50 -values were determined at elevated 2OG (~330 ·K m ; K m = 0.6 μM 30 ), elevated substrate peptide (hFX-CP 101-119 ; ~8 ·K m ; K m = 1.2 μM 30 ) or elevated Fe(II) (~14 ·K m ; K m = 1.4 μM 30 ) assay concentrations, respectively ( Table 2 ). We initially studied the potent but non-selective AspH inhibitor 2,4-PDCA ( Table 1 , entry 1) as a positive control.…”

“… 29 Cyclic peptides were designed to mimic the central non-canonical (Cys 3–4) EGFD disulfide macrocycle and were applied as stable AspH substrate analogues in kinetic experiments, the results of which suggest that AspH activity may be unusually susceptible to limiting oxygen availability and thus that AspH might be involved in the physiologically relevant hypoxic response. 30 High-throughput solid phase extraction coupled to mass spectrometry (SPE-MS) AspH inhibition assays using a stable substrate analogue were developed and employed to identify potent AspH inhibitor templates. 21 …”