Prolyl hydroxylases (PHDs) are a group of enzymes that play roles in the regulation of cellular responses to oxygen levels, primarily through their interaction with hypoxia-inducible factor (HIF). These enzymes catalyse the hydroxylation of specific proline residues within the oxygen-dependent degradation domain (ODD) of HIF, marking it for degradation under normoxic conditions. The structure and function of prolyl hydroxylases are therefore key factors in oxygen-sensing. Prolyl hydroxylases belong to the family of non-heme iron (II)- and 2-oxoglutarate-dependent dioxygenases. These share a conserved structural fold known as the double-stranded beta-helix (DSBH) fold, common in enzymes of this dioxygenase family. This fold consists of two beta sheets wrapping around a central helical core, creating a pocket that accommodates the substrate, HIF, and cofactors involved in the hydroxylation reaction. Prolyl hydroxylases consist of several conserved structural elements, including: 1) the catalytic domain, containing the active site; 2) the 2-oxoglutarate binding site, crucial for the enzymatic reaction; 3) the iron-binding motif which binds the iron(II) ion coordinated by histidine residues in the enzyme; 4) the substrate-binding site, a recognition site which accommodates the target proline residues on HIF, allowing them to be hydroxylated in an oxygen-dependent manner. Prolyl hydroxylases primarily function as oxygen sensors, playing a crucial role in regulating the stability of HIF, the transcription factor central to the cellular response to hypoxia. In hypoxic conditions (low oxygen levels), prolyl hydroxylases are catalytically less active due to the limited availability of oxygen as a substrate. As a result, hydroxylation of HIF is reduced, and the protein accumulates in the cytoplasm. Unhydroxylated HIF-α subunits translocate to the nucleus, heterodimerize with HIF-β/ARNT, and bind to hypoxia-response elements (HREs) in target genes' promoters. This leads to the upregulation of genes involved in angiogenesis, erythropoiesis, glycolysis, and other processes that help cells adapt to low oxygen conditions. Substrates of prolyl hydroxylases (PHDs) are proteins containing specific proline residues targeted for hydroxylation by these enzymes. The hydroxylation of these substrates by PHDs is a critical step in determining the stability, activity, and functions of the target proteins. PHDs also hydroxylate other proteins that are structurally related to HIF. For example, factor inhibiting HIF (FIH) hydroxylates an asparagine residue within the C-terminal transactivation domain of HIF-α, inhibiting its interaction with transcriptional coactivators. This process modulates the transcriptional activity of HIF by preventing its full transcriptional activation. Beyond their role in HIF regulation, PHDs also target collagen and other extracellular matrix proteins. Hydroxylation of proline residues within collagen provides structural stability by promoting collagen cross-linking, which is essential for tissue integrity and wound healing. Studies have identified additional proteins that can be hydroxylated by PHDs, expanding their functional roles beyond HIF regulation. One of these substrates is Ankyrin Repeat Domain 37 (ANKRD37) with hydroxylation modulating its interaction with HSP90 and affecting cellular protein folding and quality control mechanisms, whilst in another, F-Box and WD Repeat Domain Containing 7 (FBXW7), PHD-dependent hydroxylation enhances its ability to degrade hypoxia-associated proteins, contributing to hypoxic adaptation. Explore our full prolyl hydroxylases product range below and discover more, for less.