Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli

Abstract

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to gamma-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to gamma-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel gamma-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli.

FIGURE 1.

Changes in the metabolite profiles during in vitro reaction with YihU. Two-dimensional density plots of anionic metabolite profiles obtained by CE-TOFMS analysis after in vitro reaction with (right panel) or without (left panel) the YihU protein. The color intensity on the maps represent total ion count according to the scale below. The inset in the upper right box of each panel is a magnified view of the area of the rectangle at the bottom left. Arrows mark metabolites whose level changed significantly in the reaction mixture containing YihU and are labeled with metabolite m/z.

FIGURE 2.

Confirmation of in vitro enzymatic activity of YihU toward SSA by CE-TOFMS. A, selected ion electropherograms. Reactions were performed in MOPS buffer (pH 7.2) supplemented with salts and metals (“Experimental Procedures”) and using 5 mm SSA, 1 mm NADH, and YihU protein. Selected ion (anion) electropherograms show substrates (SSA and NADH) and products (unknown anion with m/z 103.04 and NAD⁺) of the reaction. Signals from each electropherogram are overlaid and show metabolites in the presence (red) and absence (blue) of YihU protein. B, proposed reaction catalyzed by YihU protein.

FIGURE 3.

MS/MS spectra of GHB (upper panel) and an unknown anion (m/z 103.04) (lower panel) obtained by CE-Q-TOFMS. Numbers adjacent to each peak indicate m/z values of their respective ions.

FIGURE 4.

Amino acid sequence alignments of YihU (P0A9V8) and its β-hydroxyacid dehydrogenase paralogs in E. coli (P0ABQ2, P77161, and Q46888) and GHBDH in A. thaliana (Q94B07). All sequences were obtained from the UniProtKB/Swiss-Prot data base. Alignments were performed using the CLUSTALW (version 1.83) multiple sequence alignments using the default parameter through the DDBJ web interface. Residues conserved in at least four proteins are highlighted in red. Conserved domains associated with putative specific functions are indicated with thick black bars.

FIGURE 5.

Survival of E. coli cells following exogenous SSA addition. Survival (ratio) indicates the ratio of colony forming unit of treated over untreated cells (0 mm). Plots indicate wild type strain (circles) and yihU knock-out strain (triangles), respectively. Values represent the average of three independent determinations and error bars indicate the mean ± S.E.

FIGURE 6.

Time course of intracellular metabolite levels in E. coli subjected to SSA treatment. Measurement of metabolites was performed by CE-TOFMS. The x axis and y axis indicate time (min) after SSA addition and metabolite level (nmol/OD/ml), respectively. The level of metabolite is displayed as amount of metabolite (nmol) in 1 ml of culture (OD₅₉₀ = 1) and represents the difference between untreated and treated samples. The open and closed circles represent the control and YihU overproducing strain, respectively. The expected YihU reaction (EC 1.1.1.61) is highlighted by a shadowed box. The data shown are from a representative dataset from three independent experiments. The other two datasets are in supplemental Fig. S5.