Metabolite profiling reveals YihU as a novel hydroxybutyrate dehydrogenase for alternative succinic semialdehyde metabolism in Escherichia coli.
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.
Saito
Natsumi
N
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017. Electronic address: natsumi@ttck.keio.ac.jp.
Robert
Martin
M
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017.
Kochi
Hayataro
H
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017.
Matsuo
Goh
G
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017.
Kakazu
Yuji
Y
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017.
Soga
Tomoyoshi
T
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017; Human Metabolome Technologies Inc., Tsuruoka, Yamagata 997-0052, Japan.
Tomita
Masaru
M
From the Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017; Human Metabolome Technologies Inc., Tsuruoka, Yamagata 997-0052, Japan.
D000429:Alcohol Oxidoreductases / Q000235:genetics; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D006884:Hydroxybutyrate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D055442:Metabolome; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012978:Sodium Oxybate / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology; D050644:Succinate-Semialdehyde Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000633:toxicity
D029968:Escherichia coli Proteins; D005680:gamma-Aminobutyric Acid; D012978:Sodium Oxybate; D000429:Alcohol Oxidoreductases; D006884:Hydroxybutyrate Dehydrogenase; C542874:YihU protein, E coli; C021398:3-hydroxyisobutyrate dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase; D050644:Succinate-Semialdehyde Dehydrogenase; C009338:succinic semialdehyde