Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

Abstract

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.

Prolyl 4-hydroxylase

“…The iron is bound in the active site by two histidine residues and an aspartate residue. The spatial orientation of these three residues around the iron is not known in P4H, though that orientation is critical for enzymatic activity (Gorres et al , 2009). This 2-His–1-carboxylate motif is common to the α-ketoglutarate-dependent, iron(II) dioxygenases.…”

“…In the halogenases, the carboxylate (Asp or Glu) is replaced by an alanine residue and a halide ion. Simply replacing the active-site Asp of human P4H with an alanine residue does not, however, endow the enzyme with halogenase activity (Gorres et al , 2009). Overall, the α-ketoglutarate-dependent dioxygenases show low sequence identity, but do share a common three-dimensional structural fold.…”

Expanding the Enzyme Universe: Accessing Non‐Natural Reactions by Mechanism‐Guided Directed Evolution

“…To further reinforce this notion, simple substitution of coordinating Asp to Ala on prolyl 4-hydroxylase, an Fe/αKG enzyme, led to an inactive enzyme. 65 Thus, despite the presumed evolutionary relationship between the two enzymes, the simplistic notion of creating a vacant coordination site for halide binding to convert a hydroxylase to halogenase only works in very special cases.…”

Toxicogenomic effect of nickel and beyond

“…Isothermal titration calorimetry analysis suggested that Ni (II) binds to ABH2 with higher affinity than Fe (II). It is likely that dioxygenases such as PHD family, JmjC-domain containing histone demethylases and ABH DNA repair enzymes use the same iron coordinating motif (His-Asp-His) to bind ferrous iron at their active sites, and Ni(II) competes with Fe(II) and replaces it at the iron-binding site (Chen et al 2010a; Gorres et al 2009). …”