Abstract
d-2-Hydroxyglutarate (d-2-HG) is a metabolite involved in many physiological metabolic processes. When d-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or d-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates d-2-HG dehydrogenase expression and responds to the presence of d-2-HG. Based on the allosteric effect of DhdR, a d-2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect d-2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of d-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages.
Highlights
D-2-Hydroxyglutarate (D-2-HG) is a metabolite involved in many physiological metabolic processes
To identify allosteric transcription factors (aTFs) that respond to D-2-HG and regulate the expression of D-2HG dehydrogenase (D2HGDH), two open reading frames (ORFs) of upstream and two ORFs of downstream of d2hgdh in bacteria containing d2hgdh were subjected to the gene occurrence profile analysis
Genes encoding GntR family transcriptional regulators, electron transfer flavoprotein (ETF), 3-phosphoglycerate dehydrogenase (SerA), carbohydrate diacid regulator (CdaR), lactate permease (LldP), and glycolate oxidase iron-sulfur subunit (GlcF) appear to be the most frequently observed in the neighborhood of d2hgdh (Supplementary Table 1)
Summary
D-2-Hydroxyglutarate (D-2-HG) is a metabolite involved in many physiological metabolic processes. DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates D-2-HG dehydrogenase expression and responds to the presence of D-2HG. D-2-HG is present at rather low levels (< 0.9 μM in the plasma of healthy humans) under physiological conditions[1,10] This suggests that organisms may have evolved specific mechanisms to respond to D-2-HG accumulation by increasing D-2-HG catabolism through enhanced D2HGDH expression[11–14]. We identify a D-2-HG catabolism regulator DhdR in Achromobacter denitrificans NBRC 15125 This aTF can repress expression of the D2HGDH-encoding gene d2hgdh, and is derepressed by D-2-HG. We combine DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology, a bead-based immunoassay, to develop a D-2-HG biosensor with high specificity and sensitivity. We use the biosensor to identify UDP-2-acetamido-2-deoxy-D-glucuronic acid (UDP-GlcNAcA) 3-dehydrogenase (WbpB) of Pseudomonas aeruginosa PAO1 as a D-2-HG anabolic enzyme that participates in the intracellular generation of D-2-HG from 2-KG
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