Active site characterization and activity of the human aspartyl (asparaginyl) ß-hydroxylase.

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the ß-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Human aspartyl (asparaginyl) hydroxylase. A multifaceted enzyme with broad intra- and extra-cellular activity.

Human aspartyl (asparaginyl) ß-hydroxylase (HAAH), a unique iron and 2-oxoglutarate dependent oxygenase, has shown increased importance as a suspected oncogenic protein. HAAH and its associated mRNA are upregulated in a wide variety of cancer types, however, the current role of HAAH in the malignant transformation of cells is unknown. HAAH is suspected to play an important role in NOTCH signaling via selective hydroxylation of aspartic acid and asparagine residues of epidermal growth factor (EGF)-like domains. HAAH hydroxylation also potentially mediates calcium signaling and oxygen sensing. In this review, we summarize the current state of understanding of the biochemistry and chemical biology of this enzyme, identify key differences from other family members, outline its broader intra- and extra-cellular roles, and identify the most promising areas for future research efforts.

Broad-spectrum catalytic metallopeptide inactivators of Zika and West Nile virus NS2B/NS3 proteases.

Flaviviruses possess a conserved protease that is vital for viral maturation. We have designed catalytic metallopeptides for inactivation of both Zika and West Nile viral proteases, and potentially other viral homologues, by irreversible target destruction and low off-target activity against host proteases. Oxidative damage promoted by metallopeptides was characterized by mass spectrometry, localized to specific active site residues, and correlated with catalyst activity.

Attenuation of West Nile Virus NS2B/NS3 Protease by Amino Terminal Copper and Nickel Binding (ATCUN) Peptides.

West Nile virus NS2B/NS3 protease (WNVP) is a viable target for the development of antiviral compounds. To that end, catalytic metallopeptides that incorporate the copper-binding ATCUN motif into either the N- or C-terminus of known WNVP targeting peptides have been developed as new families of peptide-based inhibitors. Each metallopeptide was evaluated based on its inhibitory constant (KI), time-dependent inactivation of the protein, Michaelis-Menten parameters, and the ability to oxidatively modify WNVP. Following catalytic inactivation of WNVP, sequencing by LC-MS/MS demonstrated active site residues Ser135, Thr134, and Thr132, as well as residues in the S2 binding pocket, to be modified by oxidative chemistry. Results from a DNPH-based assay to detect oxidative damage showed the formation of carbonyls in WNVP treated with metallopeptides. These results suggest that the metallopeptides are attenuating WNVP activity by irreversible oxidation of amino acids essential to substrate binding and catalysis.