Identification of Escherichia coli YgaF as an L-2-hydroxyglutarate oxidase

Abstract

YgaF, a protein of previously unknown function in Escherichia coli, was shown to possess noncovalently bound flavin adenine dinucleotide and to exhibit L-2-hydroxyglutarate oxidase activity. The inability of anaerobic, reduced enzyme to reverse the reaction by reducing the product alpha-ketoglutaric acid is explained by the very high reduction potential (+19 mV) of the bound cofactor. The likely role of this enzyme in the cell is to recover alpha-ketoglutarate mistakenly reduced by other enzymes or formed during growth on propionate. On the basis of the identified function, we propose that this gene be renamed lhgO.

FIG. 1.

Location of ygaF and regulation of the csiD-ygaF-gabDTP-csiR gene cluster. The gene that encodes YgaF is positioned downstream of and is coregulated with csiD, in part through the action of CsiR. Although located immediately upstream of the gabDTP operon, it is not involved in GABA metabolism.

FIG. 2.

Titration of YgaF with sulfite. (A) YgaF (15 μM) was titrated with 25, 50, 75, 100, 125, 150, 200, 250, 500, and 700 μM sulfite in 25 mM HEPES buffer, pH 7.0, containing 100 mM NaCl, 5 mM EDTA, 1 mM DTT, and 20% glycerol. (B) Absorbance change (ΔAbs) at 450 nm versus the concentration of free sulfite.

FIG. 3.

Analysis of YgaF flavin reduction potential. (A) An anaerobic mixture of YgaF and MB (10 μM each) was titrated with sodium dithionite while monitoring the absorption spectrum. The changes in the relative concentrations of the reduced and oxidized forms of MB were used to deduce the system reduction potential (E) for each condition. (B) Correlation of E with the log(FAD_ox/FAD_red) of YgaF.

FIG. 4.

Titration of anaerobic YgaF with l-2-hydroxyglutarate. (A) Anaerobic YgaF (47 μM in 25 mM HEPES, pH 8.2, containing 100 mM NaCl, 5 mM EDTA, 1 mM DTT, and 20% glycerol) was adjusted to contain 19, 38, 57, 95, 133, and 171 μM l-2-hydroxyglutarate. (B) Fractional change in absorbance at 450 nm as a function of the concentration of free substrate in the solution.

FIG. 5.

Time course of α-ketoglutarate (αKG) production for the reaction of YgaF with the two enantiomers of 2-hydroxyglutarate. YgaF (1 μM) was incubated with d-2-hydroxyglutarate (300 μM) in 25 mM HEPES buffer containing 100 mM NaCl, 1 mM DTT, and 20% glycerol at pH 8.2 at 25°C. Aliquots of 970 μl were collected from the reaction mixture at 1, 3, 5, 10, and 25 min and treated with OPDA (square symbols). The remaining reaction mixture was separated into two equal portions. Additional d-2-hydroxyglutarate (300 μM) was added to one portion (triangle symbols), and l-2-hydroxyglutarate (300 μM) was added to the other (circles). Aliquots of 970 μl were collected from both reactions at 1, 3, 5, 10, 20, and 40 min and treated with OPDA.

FIG. 6.

The reaction catalyzed by YgaF.