ABSTRACT
AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1-3, 2-4, 5-6 disulfide bonding pattern; an unexpected Cys3-4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
Subject(s)
Calcium-Binding Proteins/chemistry , Membrane Proteins/chemistry , Mixed Function Oxygenases/chemistry , Muscle Proteins/chemistry , Amino Acid Sequence , Asparagine/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Catalytic Domain , Crystallography , Disulfides/chemistry , Disulfides/metabolism , Epidermal Growth Factor/metabolism , Ferrous Compounds/chemistry , Ferrous Compounds/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Protein ConformationABSTRACT
γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate and Fe(II) dependent oxygenase that catalyses an essential step during carnitine biosynthesis in animals. BBOX is inhibited by ejection of structural zinc by a set of selenium containing analogues. Previous structural analyses indicated that an undisrupted N-terminal zinc binding domain of BBOX is required for catalysis. Ebselen is a relatively potent BBOX inhibitor, an observation which may in part reflect its cardioprotective properties.
Subject(s)
Azoles/pharmacology , Enzyme Inhibitors/pharmacology , Organoselenium Compounds/pharmacology , Zinc/metabolism , gamma-Butyrobetaine Dioxygenase/antagonists & inhibitors , Catalysis , Humans , Isoindoles , Models, Molecular , Oxidation-Reduction , Protein Binding , Protein Conformation , Structure-Activity Relationship , gamma-Butyrobetaine Dioxygenase/metabolismABSTRACT
γ-Butyrobetaine hydroxylase (BBOX) is a 2-oxoglutarate dependent oxygenase that catalyzes the final hydroxylation step in the biosynthesis of carnitine. BBOX was shown to catalyze the oxidative desymmetrization of achiral N,N-dialkyl piperidine-4-carboxylates to give products with two or three stereogenic centers.
Subject(s)
Carboxylic Acids/metabolism , gamma-Butyrobetaine Dioxygenase/metabolism , Biocatalysis , Carboxylic Acids/chemistry , Carnitine/biosynthesis , Carnitine/chemistry , Catalytic Domain , Oxidation-Reduction , Piperidines/chemistry , Stereoisomerism , gamma-Butyrobetaine Dioxygenase/chemistryABSTRACT
2-Oxoglutarate and iron dependent oxygenases have potential for the stereoselective hydroxylation of amino acids and related compounds. The biochemical and kinetic properties of recombinant γ-butyrobetaine hydroxylase from human and Pseudomonas sp. AK1 were compared. The results reveal differences between the two BBOXs, including in their stimulation by ascorbate. Despite their closely related sequences, the two enzymes also display different substrate selectivities, including for the production of (di)hydroxylated betaines, implying use of engineered BBOXs for biocatalytic purposes may be productive.
Subject(s)
Pseudomonas/enzymology , gamma-Butyrobetaine Dioxygenase/metabolism , Biocatalysis , Humans , Models, Molecular , Molecular Structure , Substrate Specificity , gamma-Butyrobetaine Dioxygenase/chemistryABSTRACT
Carnitine is essential for fatty acid metabolism, but is associated with both health benefits and risks, especially heart diseases. We report the identification of potent, selective and cell active inhibitors of γ-butyrobetaine hydroxylase (BBOX), which catalyses the final step of carnitine biosynthesis in animals. A crystal structure of BBOX in complex with a lead inhibitor reveals that it binds in two modes, one of which adopts an unusual 'U-shape' conformation stabilised by inter- and intra-molecular π-stacking interactions. Conformational changes observed on binding of the inhibitor to BBOX likely reflect those occurring in catalysis; they also rationalise the inhibition of BBOX by high levels of its substrate γ-butyrobetaine (GBB), as observed both with isolated BBOX protein and in cellular studies.
ABSTRACT
A convenient method for the synthesis of fluoromethylated carnitine biosynthesis intermediates, i.e. fluorinated derivatives of γ-butyrobetaine and trimethyllysine, is described. The fluoromethylated probes were useful in both in vitro and cell based assays employing (19)F NMR and LC-MS analyses.
Subject(s)
Carnitine/chemistry , Fluorine/chemistry , Cell Line , Escherichia , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Humans , Methylation , Molecular StructureABSTRACT
Fluoride assays for oxygenases: The 2-oxoglutarate-dependent oxygenase BBOX catalyses the final step in carnitine biosynthesis and is a medicinal chemistry target. We report that BBOX can hydroxylate fluorinated substrates analogues with subsequent release of a fluoride ion, thereby enabling an efficient fluorescence-based assay.
Subject(s)
Fluorides/analysis , gamma-Butyrobetaine Dioxygenase/analysis , gamma-Butyrobetaine Dioxygenase/metabolism , Biocatalysis , Fluorescence , Humans , Hydrocarbons, Fluorinated/chemistry , Hydrocarbons, Fluorinated/metabolism , Magnetic Resonance Spectroscopy , Molecular Structure , ProtonsABSTRACT
Phytanoyl-CoA hydroxylase (PAHX) catalyzes an important step in the metabolism of the fatty acid side chain of chlorophyll. PHYHD1 exists in three isoforms and is the closest human homologue of PAHX. We show that like PAHX, the PHYHD1A but likely not the PHYHD1B/C isoforms, is a functional Fe(II) and 2-oxoglutarate (2OG) dependent oxygenase. Crystallographic and biochemical analyses reveal that PHYHD1A has the double-stranded ß-helix fold and Fe(II) and cosubstrate binding residues characteristic of the 2-oxoglutarate dependent oxygenases and catalyzes the conversion of 2-oxoglutarate to succinate and CO(2) in an iron-dependent manner. However, PHYHD1A did not couple 2OG turnover to the hydroxylation of acyl-coenzyme A derivatives that are substrates for PAHX, implying that it is not directly involved in phytanoyl coenzyme-A metabolism.
Subject(s)
Mixed Function Oxygenases/chemistry , Oxygenases/chemistry , Crystallography, X-Ray , Humans , Iron/chemistry , Mixed Function Oxygenases/genetics , Oxygenases/genetics , Phosphorylation , Protein Processing, Post-Translational , Protein Structure, SecondaryABSTRACT
The final step in carnitine biosynthesis is catalyzed by γ-butyrobetaine (γBB) hydroxylase (BBOX), an iron/2-oxoglutarate (2OG) dependent oxygenase. BBOX is inhibited by trimethylhydrazine-propionate (THP), a clinically used compound. We report structural and mechanistic studies on BBOX and its reaction with THP. Crystallographic and sequence analyses reveal that BBOX and trimethyllysine hydroxylase form a subfamily of 2OG oxygenases that dimerize using an N-terminal domain. The crystal structure reveals the active site is enclosed and how THP competes with γBB. THP is a substrate giving formaldehyde (supporting structural links with histone demethylases), dimethylamine, malonic acid semi-aldehyde, and an unexpected product with an additional carbon-carbon bond resulting from N-demethylation coupled to oxidative rearrangement, likely via an unusual radical mechanism. The results provide a basis for development of improved BBOX inhibitors and may inspire the discovery of additional rearrangement reactions.
