Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase

Pircher, Haymo; Grafenstein, Susanne von; Diener, Thomas; Metzger, Christina; Albertini, Eva; Taferner, Andrea; Unterluggauer, Hermann; Krämer, Christian; Liedl, Klaus R.; Jansen‐Dürr, Pidder

doi:10.1074/jbc.m114.609305

Cited by 33 publications

(50 citation statements)

References 21 publications

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“…(A) ODx activity of mFAHD1: within the accuracy of a photometric assay [7], v max ranges from 0.18 to 0.24 μmol/min/mg, and K M ranges from 8 to 78.0 μM, similar to hFAHD1 [6,23]. Within the given range, our best-model fit found v max = 0.21 μmol/min/mg and K M = 42.8 μM (see Table 2).…”

Section: Figure 4 Michaelis-menten Kinetics Of Odx and Aph Activitiementioning

confidence: 74%

“…Polyclonal antisera against FAHD1 were raised against recombinant untagged hFAHD1 protein and His6/S-doubl e-tagged mFAHD1 protein, both produced in E. coli [23]. Recombinant protein was purified from E. coli using a series of three subsequent chromatographic steps as described [7,23]. Approximately 4 mg of pure recombinant protein was used by a commercial supplier (Biogenes, Berlin, Germany) to produce specific antisera which were shipped to our laboratory.…”

Section: Antibodiesmentioning

confidence: 99%

“…FAHD1-specific antibodies were obtained from raw sera by protein G affinity chromatography and gel filtration, followed by a second affinity purification using an affinity resin consisting of sepharose beads with immobilized purified antigen. Rabbit monoclonal anti-mouse FAHD1 antibodies, including RabMab 27-1 [23] were raised against affinity-purified recombinant mFAHD1 protein produced in E. coli. Four milligrams of purified His6/S-double-tagged mFAHD1 [7] were used by a commercial supplier (Epitomics Inc, Burlingame, U.S.A.) to produce several hybridoma clones, which were delivered to our lab.…”

Section: Antibodiesmentioning

confidence: 99%

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Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

et al. 2020

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FAH domain containing protein 1 (FAHD1) is a mammalian mitochondrial protein, displaying bifunctionality as acylpyruvate hydrolase (ApH) and oxaloacetate decarboxylase (ODx) activity. We report the crystal structure of mouse FAHD1 and structural mapping of the active site of mouse FAHD1. Despite high structural similarity with human FAHD1, a rabbit monoclonal antibody (RabMab) could be produced that is able to recognize mouse FAHD1, but not the human form, whereas a polyclonal antibody recognized both proteins. Epitope mapping in combination with our deposited crystal structures revealed that the epitope overlaps with a reported SIRT3 deacetylation site in mouse FAHD1.culture was grown in 1000 ml NZCYM medium, containing the respective selective antibiotics. At 37 • C the bacteria were amplified to an optical density of 0.4 at 600 nm. Protein expression was induced by the addition of 500 μM isopropyl-1-thio-β-d-galactopyranoside (IPTG) and incubation was continued for 4 h at 37 • C. Bacteria were harvested via centrifugation and stored at −70 • C. Enzyme purificationEnzyme purification of His6/S-double-tagged proteins followed a defined protocol [7]. Recombinant protein was extracted via a three-step purification strategy involving metal affinity chromatography (Ni-NTA), anion exchange chromatography, and gel filtration (SEC). Fractions containing FAHD1 were pooled, concentrated and stored at −70 • C. Gel electrophoresis of two preparations followed by silver staining verified the protein's homogeneity; contaminations were barely visible (<<100 ng/μl).Recombinant mouse FAHD1 (mFAHD1) of concentration in between 1.5 and 2.0 mg/ml (Vivaspin 10 MWCO) was sent for high throughput screening at the HTX laboratory (EMBL, Grenoble). Native mFAHD1 and mFAHD1 supplemented with 1 mM oxalate were screened for crystallization, and hits were obtained in various conditions. A total of 68 crystals were harvested by laser photoablation, cryo-cooled using the CrystalDirect Robot [8,9], and screened for diffraction. Best diffracting crystals of mFAHD1 grew from 25% (w/v) PEG 3350, 0.2 M MgCl 2 and 0.1 M Bis/Tris pH 5.5 and for the oxalate bound protein from 20% (w/v) PEG 4000, 0.05 M MgCl 2 and 0.1 M MES pH 5.5. As the crystals were small needles with volumes between 10 −6 and 10 −5 mm 3 a beam diameter of 15 μm was selected [10]. X-ray diffraction data were collected by the autonomous European Synchrotron Radiation Facility (ESRF) beamline using automatic protocols for the location and optimal centring of crystals [14]. Strategy calculations accounted for flux and crystal volume in the parameter prediction for complete datasets. All data were processed using the automatic pipelines at the ESRF [15]. Statistical analysisKinetic profiles of recombinant protein were obtained with three independent batches of protein preparations (n=3). Data analysis was performed using GraphPad Prism version 5 (www.graphpad.com). Presented data are the mean and standard deviation within 95% confidence interval.

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Section: Figure 4 Michaelis-menten Kinetics Of Odx and Aph Activitiementioning

confidence: 74%

Section: Antibodiesmentioning

confidence: 99%

Section: Antibodiesmentioning

confidence: 99%

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Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

et al. 2020

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“…In a recent study to further investigate the cellular role of FAHD1, molecular modeling was applied to search for a close structural match between the catalytic domain of FAHD1 and other members of the FAH family [9]. This process yielded the prokaryotic enzyme Cg1458 as a promising candidate, previously identified as a soluble ODx [2].Subsequent in vitro analysis of purified recombinant human FAHD1 confirmed that it indeed exhibits ODx activity.…”

Section: Editorialmentioning

confidence: 99%

“…It can be expected that studies with available FAHD1-/-mice [9] will shed light on the enigmatic role of the newly identified ODx FAHD1 and help to elucidate its functional interaction with other metabolic pathways. Fumarylacetoacetate hydrolase (FAH) domain-containing protein 1 (FAHD1) is found to play a key role in central metabolism, in particular at the intersection of catabolic pathways and the tricarboxylic acid cycle (TCA).…”

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confidence: 99%

The FAH Fold Meets the Krebs Cycle

Jansen-Duerr¹,

Pircher²,

Weiss³

2016

Mol Enzyme Drug Targ

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EditorialThe Krebs (or tricarboxylic acid, TCA) cycle forms a central junction in aerobic metabolism, being connectedto glycolysis, gluconeogenesis, fatty acid metabolism and amino acid metabolism ( Figure 1). Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate to form oxaloacetate, an important so-called anaplerotic reaction to provide a sufficient level of metabolites for the TCA cycle. The breakdown of oxaloacetate to pyruvate by oxaloacetate decarboxylases (ODx) is mainly known from prokaryotic organisms where two main variants of ODx enzymes are known: a membrane-bound variant that depends on sodium and biotin [1] and a soluble variant that depends on divalent cations [2]. Far less is known about ODx activity in mammalian organisms, apart from the promiscuous activity of some metabolic enzymes [3].Many decades ago several reports about an ODx enzyme purified from extracts of rat liver mitochondria surfaced [4,3], but curiously enough the identity of such an enzyme was never clarified.Until recently, members of the FAH family of enzymes were not known to be connected to the TCA cycle in any way. The eponymous FAH enzyme acts as a fumarylacetoacetate hydrolase, catalyzing the final step of the tyrosine breakdown [5]. Loss of this function causes hereditary tyrosinemia type I (HTI) in humans [6].The prokaryotic realm provides a perplexingly large number of FAH type enzymes which, in spite of the structural similarity of their catalytic domains, collectively referred to as the FAH fold, catalyze a wide range of reactions [7]. They comprise hydrolases, isomerases, decarboxylases, hydratases and dehydratases, demonstrating the versatility of the FAH fold. Besides FAH itself, the only other members of the FAH superfamily present in mammalian organisms are FAHD1 (fumarylacetoacetate hydrolase domain containing protein 1) and FAHD2. Classification of their function has proven difficult due to the aforementioned spectrum of FAH fold activities. In a first study, the comparison of FAHD1's primary sequence with prokaryotic members of the FAH family led to the observation that FAHD1 exhibits acylpyruvate hydrolaseThis activity is so far not associated with mammalian metabolism, however. In a recent study to further investigate the cellular role of FAHD1, molecular modeling was applied to search for a close structural match between the catalytic domain of FAHD1 and other members of the FAH family [9]. This process yielded the prokaryotic enzyme Cg1458 as a promising candidate, previously identified as a soluble ODx [2].Subsequent in vitro analysis of purified recombinant human FAHD1 confirmed that it indeed exhibits ODx activity. Interestingly, the enzyme kinetics of FAHD1 closely resemble some of the values reported for an unidentified mammalian ODx in earlier years [4,3]. The mutation of key residues located within the catalytic domain, including a catalytic loop, resulted in abrogated or reduced enzymatic activity.As already observed in an earlier study [8], FAHD1 is located in mitochondria, showing high...

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The mitochondrial enzyme FAHD1 regulates complex II activity in breast cancer cells and is indispensable for basal BT‐20 cells in vitro

et al. 2022

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The mitochondrial enzyme fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) was identified to be upregulated in breast cancer tissues. Here, we show that FAHD1 is indispensable for the survival of BT-20 cells, representing the basal breast cancer cell type. A lentiviral knock-down of FAHD1 in the breast cancer cell lines MCF-7 and BT-20 results in lower succinate dehydrogenase (complex II) activity. In luminal MCF-7 cells, this leads to reduced proliferation when cultured in medium containing only glutamine as the carbon source. Of note, both cell lines show attenuated protein levels of the enzyme glutaminase (GLS) which activates programmed cell death in BT-20. These findings demonstrate that FAHD1 is crucial for the functionality of complex II in breast cancer cells and acts on glutaminolysis in the mitochondria.

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Identification of FAH Domain-containing Protein 1 (FAHD1) as Oxaloacetate Decarboxylase

Cited by 33 publications

References 21 publications

Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

Structural and functional comparison of fumarylacetoacetate domain containing protein 1 in human and mouse

The FAH Fold Meets the Krebs Cycle

The mitochondrial enzyme FAHD1 regulates complex II activity in breast cancer cells and is indispensable for basal BT‐20 cells in vitro

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