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► Structural features of 2OG oxygenases involved in substrate recognition are analyzed. ► Crystallographic studies reveal the versatility of the jelly roll fold in substrate ...binding. ► Defined structural regions that interact with substrate(s) are biased by fold topology. ► The utility of the enzyme–substrate structures for engineering and selective inhibition are discussed.
2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases catalyze two-electron oxidations of a range of small and large molecule substrates, including proteins/peptides/amino acids, nucleic acids/bases, and lipids, as well as natural products including antibiotics and signaling molecules. 2OG oxygenases employ variations of a core double-stranded β-helix (DSBH; a.k.a. jelly-roll, cupin or jumonji C (JmjC)) fold to enable binding of Fe(II) and 2OG in a subfamily conserved manner. The topology of the DSBH limits regions directly involved in substrate binding: commonly the first, second and eighth strands, loops between the second/third and fourth/fifth DSBH strands, and the N-terminal and C-terminal regions are involved in primary substrate, co-substrate and cofactor binding. Insights into substrate recognition by 2OG oxygenases will help to enable selective inhibition and bioengineering studies.
The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates ...including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry- and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.
In 2007, a genome wide association study identified a SNP in intron one of the gene encoding human FTO that was associated with increased body mass index. Homozygous risk allele carriers are on ...average three kg heavier than those homozygous for the protective allele. FTO is a DNA/RNA demethylase, however, how this function affects body weight, if at all, is unknown. Here we aimed to pharmacologically inhibit FTO to examine the effect of its demethylase function in vitro and in vivo as a first step in evaluating the therapeutic potential of FTO. We showed that IOX3, a known inhibitor of the HIF prolyl hydroxylases, decreased protein expression of FTO (in C2C12 cells) and reduced maximal respiration rate in vitro. However, FTO protein levels were not significantly altered by treatment of mice with IOX3 at 60 mg/kg every two days. This treatment did not affect body weight, or RER, but did significantly reduce bone mineral density and content and alter adipose tissue distribution. Future compounds designed to selectively inhibit FTO's demethylase activity could be therapeutically useful for the treatment of obesity.
Significance The Fe(II)- and 2-oxoglutarate (2OG)-dependent hypoxia-inducible transcription factor prolyl-hydroxylases play a central role in human oxygen sensing and are related to other ...prolyl-hydroxylases involved in eukaryotic collagen biosynthesis and ribosomal modification. The finding that a PHD-related prolyl-hydroxylase in Pseudomonas spp. regulates pyocyanin biosynthesis supports prokaryotic origins for the eukaryotic prolyl-hydroxylases. The identification of the switch I loop of elongation factor Tu (EF-Tu) as a Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) substrate provides evidence of roles for 2OG oxygenases in both translational and transcriptional regulation. A structure of the PPHD:EF-Tu complex, the first to the authors' knowledge of a 2OG oxygenase with its intact protein substrate, reveals that major conformational changes occur in both PPHD and EF-Tu and will be useful in the design of new prolyl-hydroxylase inhibitors.
2-Oxoglutarate-dependent nucleic acid demethylases are of biological interest because of their roles in nucleic acid repair and modification. Although some of these enzymes are linked to physiology, ...their regulatory roles are unclear. Hence, there is a desire to develop selective inhibitors for them; we report studies on AlkB, which reveal it as being amenable to selective inhibition by small molecules. Dynamic combinatorial chemistry linked to mass spectrometric analyses (DCMS) led to the identification of lead compounds, one of which was analyzed by crystallography. Subsequent structure-guided studies led to the identification of inhibitors of improved potency, some of which were shown to be selective over two other 2OG oxygenases. The work further validates the use of the DCMS method and will help to enable the development of inhibitors of nucleic acid modifying 2OG oxygenases both for use as functional probes and, in the longer term, for potential therapeutic use.
Abstract
ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N6-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and ...plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH566–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.
The use of β-lactam antibiotics is compromised by resistance, which is provided by β-lactamases belonging to both metallo (MBL)- and serine (SBL)-β-lactamase subfamilies. The rhodanines are one of ...very few compound classes that inhibit penicillin-binding proteins (PBPs), SBLs and, as recently reported, MBLs. Here, we describe crystallographic analyses of the mechanism of inhibition of the clinically relevant VIM-2 MBL by a rhodanine, which reveal that the rhodanine ring undergoes hydrolysis to give a thioenolate. The thioenolate is found to bind via di-zinc chelation, mimicking the binding of intermediates in β-lactam hydrolysis. Crystallization of VIM-2 in the presence of the intact rhodanine led to observation of a ternary complex of MBL, a thioenolate fragment and rhodanine. The crystallographic observations are supported by kinetic and biophysical studies, including (19)F NMR analyses, which reveal the rhodanine-derived thioenolate to be a potent broad-spectrum MBL inhibitor and a lead structure for the development of new types of clinically useful MBL inhibitors.
Post-translational ribosomal protein hydroxylation is catalyzed by 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases, and occurs in prokaryotes and eukaryotes. OGFOD1 catalyzes trans-3 ...prolyl hydroxylation at Pro62 of the small ribosomal subunit protein uS12 (RPS23) and is conserved from yeasts to humans. We describe crystal structures of the human uS12 prolyl 3-hydroxylase (OGFOD1) and its homolog from Saccharomyces cerevisiae (Tpa1p): OGFOD1 in complex with the broad-spectrum 2OG oxygenase inhibitors; N-oxalylglycine (NOG) and pyridine-2,4-dicarboxylate (2,4-PDCA) to 2.1 and 2.6 Å resolution, respectively; and Tpa1p in complex with NOG, 2,4-PDCA, and 1-chloro-4-hydroxyisoquinoline-3-carbonylglycine (a more selective prolyl hydroxylase inhibitor) to 2.8, 1.9, and 1.9 Å resolution, respectively. Comparison of uS12 hydroxylase structures with those of other prolyl hydroxylases, including the human hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs), reveals differences between the prolyl 3- and prolyl 4-hydroxylase active sites, which can be exploited for developing selective inhibitors of the different subfamilies.
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•Crystal structures of human OGFOD1 in complex with inhibitors are presented•The OGFOD1 and PHDs likely share a common ancestor•The OGFOD1 active site is comparable with hypoxia-inducible factor prolyl hydroxylase•Differences between OGFOD1 and PHD2 can be exploited for inhibitor design
Horita et al. present structures of the first prolyl 3-hydroxylase from humans, OGFOD1, in complex with small-molecule inhibitors. The results shed light on the catalytic mechanisms and evolution of the OGFOD1 subfamily and the related “oxygen-sensing” hypoxia-inducible factor trans-P4Hs (PHDs).
N6-Methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m6A in ...single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC50, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m6A binding pocket of ALKBH5 and the key residues involved in m6A recognition using mutagenesis and ITC binding experiments.
Background: ALKBH5 catalyzes demethylation of m6A single-stranded RNA (ssRNA).
Results: ALKBH5 structures reveal the structural basis of its substrate selectivity and inhibition by citrate.
Conclusion: ALKBH5 specifically binds to and demethylates m6A ssDNA/ssRNA. Citrate is a modest inhibitor of ALKBH5.
Significance: This study provides insights into the molecular mechanism of ALKBH5 as an m6A ssRNA demethylase and will facilitate the design of selective inhibitors.
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