Metabolite Profiling Reveals YihU as a Novel Hydroxybutyrate Dehydrogenase for Alternative Succinic Semialdehyde Metabolism in Escherichia coli
2009; Elsevier BV; Volume: 284; Issue: 24 Linguagem: Inglês
10.1074/jbc.m109.002089
ISSN1083-351X
AutoresNatsumi Saito, Martin Robert, Hayataro Kochi, Goh Matsuo, Yuji Kakazu, Tomoyoshi Soga, Masaru Tomita,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoThe 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 γ-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 γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. 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 γ-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 γ-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel γ-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli. The search for novel enzymes is important to better our understanding of the metabolic systems of the cell. Although computational tools can be used to functionally annotate enzymes based on sequence homology, gene structure and expression, and prediction of enzyme-like domains, the identification of the exact physiological substrates remains difficult when sequence similarity to known enzymes is low (<60%) and requires experimental confirmation (1Rost B. J. Mol. Biol. 2002; 318: 595-608Crossref PubMed Scopus (294) Google Scholar, 2Tian W. Skolnick J. J. Mol. Biol. 2003; 333: 863-882Crossref PubMed Scopus (306) Google Scholar). Consequently, many gaps remain in metabolic pathways even in the model microorganism Escherichia coli (3Reed J.L. Vo T.D. Schilling C.H. Palsson B.O. Genome Biol. 2003; 4: R54Crossref PubMed Google Scholar, 4Chen L. Vitkup D. Trends Biotechnol. 2007; 25: 343-348Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Moreover, the identification of dispensable enzymatic activities, such as metabolic bypass pathways or the characterization of enzymes that are expressed only under specific physiological conditions, is particularly challenging. The β-hydroxyacid dehydrogenase enzyme family is a structurally conserved group of enzymes that include β-hydroxyisobutyrate dehydrogenase, 6-phosphogluconate dehydrogenase, and numerous uncharacterized homologs (5Hawes J.W. Harper E.T. Crabb D.W. Harris R.A. FEBS Lett. 1996; 389: 263-267Crossref PubMed Scopus (37) Google Scholar, 6Njau R.K. Herndon C.A. Hawes J.W. Chem. Biol. Interact. 2001; 130–132: 785-791Crossref PubMed Scopus (26) Google Scholar). This enzyme family contains well conserved domains in its sequence that include a N-terminal Rossmann-fold characteristic of a dinucleotide binding site, a well defined sequence at the substrate binding site, and a conserved lysine residue proposed as a critical catalytic residue. This last specific structural feature has been proposed based on site-directed mutagenesis and x-ray crystal structures (6Njau R.K. Herndon C.A. Hawes J.W. Chem. Biol. Interact. 2001; 130–132: 785-791Crossref PubMed Scopus (26) Google Scholar, 7Adams M.J. Ellis G.H. Gover S. Naylor C.E. Phillips C. Structure. 1994; 2: 651-668Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The E. coli K12 proteome appears to contain four β-hydroxyacid dehydrogenase paralogs. The product of the glxR gene has been identified as tartronate semialdehyde reductase, catalyzing the NAD+-dependent oxidation of d-glycerate and the NADH-dependent reduction of tartronate semialdehyde (8Njau R.K. Herndon C.A. Hawes J.W. J. Biol. Chem. 2000; 275: 38780-38786Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This enzyme plays a role in allantoin utilization under anaerobic conditions in E. coli (9Cusa E. Obradors N. Baldomà L. Badía J. Aguilar J. J. Bacteriol. 1999; 181: 7479-7484Crossref PubMed Google Scholar). However, the function of the other three representatives of the family remains unknown. Under aerobic conditions in E. coli, γ-aminobutyrate (GABA) 2The abbreviations used are: GABAγ-aminobutyric acidGHBγ-hydroxybutyric acidSSAsuccinic semialdehydeCE-TOFMScapillary electrophoresis time-of-flight mass spectrometryMES2-(N-morpholino)ethansulfonic acidMOPS3-(N-morpholino)propanesulfonic acidGHBDHγ-hydroxybutyrate dehydrogenaseESIelectrospray ionization.2The abbreviations used are: GABAγ-aminobutyric acidGHBγ-hydroxybutyric acidSSAsuccinic semialdehydeCE-TOFMScapillary electrophoresis time-of-flight mass spectrometryMES2-(N-morpholino)ethansulfonic acidMOPS3-(N-morpholino)propanesulfonic acidGHBDHγ-hydroxybutyrate dehydrogenaseESIelectrospray ionization. is metabolized via GABA transaminase (EC 2.6.1.19) (10Bartsch K. von Johnn-Marteville A. Schulz A. J. Bacteriol. 1990; 172: 7035-7042Crossref PubMed Google Scholar) and oxidized to succinate by at least two different succinic semialdehyde dehydrogenases (EC 1.2.1.16 and EC 1.2.1.24) (11Donnelly M.I. Cooper R.A. Eur. J. Biochem. 1981; 113: 555-561Crossref PubMed Scopus (36) Google Scholar, 12Marek L.E. Henson J.M. J. Bacteriol. 1988; 170: 991-994Crossref PubMed Google Scholar), and then further metabolized in the tricarboxylic acid cycle. In some animals (13Andriamampandry C. Siffert J.C. Schmitt M. Garnier J.M. Staub A. Muller C. Gobaille S. Mark J. Maitre M. Biochem. J. 1998; 334: 43-50Crossref PubMed Scopus (26) Google Scholar), plants (14Breitkreuz K.E. Allan W.L. Van Cauwenberghe O.R. Jakobs C. Talibi D. Andre B. Shelp B.J. J. Biol. Chem. 2003; 278: 41552-41556Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and bacterial species (15Valentin H.E. Zwingmann G. Schönebaum A. Steinbüchel A. Eur. J. Biochem. 1995; 227: 43-60Crossref PubMed Scopus (66) Google Scholar, 16Wolff R.A. Kenealy W.R. Protein Expr. Purif. 1995; 6: 206-212Crossref PubMed Scopus (10) Google Scholar), γ-hydroxybutyrate (GHB) can be produced during GABA catabolism through the reduction of succinic semialdehyde (SSA) under anaerobic conditions. A γ-hydroxybutyrate dehydrogenase (GHBDH) was recently identified in Arabidopsis thaliana (14Breitkreuz K.E. Allan W.L. Van Cauwenberghe O.R. Jakobs C. Talibi D. Andre B. Shelp B.J. J. Biol. Chem. 2003; 278: 41552-41556Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Interestingly, the Arabidopsis enzyme does not show significant homology with known GHBDHs, however, its sequence exhibits similarity to several dehydrogenases including β-hydroxyacid dehydrogenases and 6-phosphogluconate dehydrogenases. However, the existence of an equivalent of the GHBDH reaction and an alternative reductive pathway for GABA metabolism in E. coli is still unreported. γ-aminobutyric acid γ-hydroxybutyric acid succinic semialdehyde capillary electrophoresis time-of-flight mass spectrometry 2-(N-morpholino)ethansulfonic acid 3-(N-morpholino)propanesulfonic acid γ-hydroxybutyrate dehydrogenase electrospray ionization. γ-aminobutyric acid γ-hydroxybutyric acid succinic semialdehyde capillary electrophoresis time-of-flight mass spectrometry 2-(N-morpholino)ethansulfonic acid 3-(N-morpholino)propanesulfonic acid γ-hydroxybutyrate dehydrogenase electrospray ionization. We have previously developed a screening method, based on in vitro assays in combination with metabolite profiling by capillary electrophoresis-mass spectrometry (CE-MS), to discover novel enzymatic activities (17Saito N. Robert M. Kitamura S. Baran R. Soga T. Mori H. Nishioka T. Tomita M. J. Proteome Res. 2006; 5: 1979-1987Crossref PubMed Scopus (68) Google Scholar). We hereby refer to this method as Metabolic Enzyme and Reaction discovery by Metabolite profile Analysis and reactant IDentification (MERMAID). Using this method, the enzymatic activity of any uncharacterized protein can be tested in an unbiased way by monitoring changes in a complex metabolite mixture that are induced by the test protein. This can allow to directly determine the substrate(s) and/or product(s) of the reaction without designing specific assays. Compounds whose levels specifically decrease following incubation with a protein are likely substrates, whereas metabolites whose level increase during the incubation are likely products of the reaction. In this study, we screened the E. coli YihU protein using the MERMAID approach and observed that it displays reductase activity toward short chain aldehydes, predominantly toward SSA. This activity differs from that of the known β-hydroxyacid dehydrogenases. We further demonstrate the presence of an alternative reaction for SSA catabolism leading to the production of GHB in E. coli. Histidine-tagged recombinant proteins were purified from ASKA clones (A Complete Set of E. coli K12 ORF Archive) (18Kitagawa M. Ara T. Arifuzzaman M. Ioka-Nakamichi T. Inamoto E. Toyonaga H. Mori H. DNA Res. 2005; 12: 291-299Crossref PubMed Scopus (1036) Google Scholar). Each of the full-length open reading frames is cloned in an archive expression vector pCA24N (GenBankTMAB052891) containing a His6 tag at the amino-terminal of the open reading frame. Recombinant proteins were produced in E. coli AG1 cells (Stratagene, La Jolla, CA) and purified using cobalt-based immobilized TALON metal affinity chromatography resins with a gravity-flow column (Clontech, Palo Alto, CA) according to the protocol provided by the manufacturer. Finally, the proteins were eluted from the column using 50 mm sodium phosphate buffer (pH 7.0) containing 150 mm NaCl and 200 mm imidazole. The protein solution was ultrafiltrated with a 10,000 nominal molecular weight limit filter (Millipore, Billerica, MA) to exchange buffer to an imidazole-free sodium phosphate buffer. The enriched protein solution supplemented with 30% of glycerol was stored at −30 °C until use. The concentration of purified proteins was determined using a protein assay reagent (Bio-Rad) using bovine serum albumin as a standard and purity was estimated by SDS-PAGE. Size exclusion chromatography was performed using a Bio-Silect SEC-250-5 column (300 × 7.8 mm) (Bio-Rad) equilibrated with 100 mm sodium phosphate buffer (pH 7.0) containing 150 mm NaCl and using an Agilent 1100 series purification system (Agilent Technologies Inc.). Fractions were collected at a flow rate of 0.5 ml/min. Calibration was performed using the gel filtration molecular weight standards from Bio-Rad. Unless otherwise stated, spectrophotometric dehydrogenase assays were performed at 37 °C in 100 μl of reaction mixture containing 5 mm substrate and 1 μg of purified protein in 100 mm MOPS-KOH buffer (pH 7.2) and 1 mm NADH (in the reductive direction) or 100 mm Tris-HCl buffer (pH 8.8) and 1 mm β-NAD+ (in the oxidative direction) together with 10 mm MgCl2, 10 mm KCl, 1 mm MnCl2. Reactions were initiated by adding substrate, and monitored by measuring the change in NADH absorbance at 340 nm. Specific activities were calculated from the rate of change in NADH amount (μmol/min) per mg of protein used. All spectrophotometric assays were performed using a SpectraMax Plus microplate spectrophotometer (Molecular Devices). Confirmation of YihU activity on GHB was performed using GHB synthesized from alkaline hydrolysis of γ-butyrolactone (19Marvel C.S. Birkhimer E.R. J. Am. Chem. Soc. 1929; 51: 260-262Crossref Scopus (17) Google Scholar). Metabolomics-based in vitro enzyme screening was performed as described previously (17Saito N. Robert M. Kitamura S. Baran R. Soga T. Mori H. Nishioka T. Tomita M. J. Proteome Res. 2006; 5: 1979-1987Crossref PubMed Scopus (68) Google Scholar) with some modifications. Reactions were performed in 20 mm Tris-HCl buffer (pH 7.2), 10 mm MgCl2, 10 mm KCl, 1 mm MnCl2, with a metabolite mixture prepared from yeast extract (BD Biosciences) supplemented with 200 μm of the following general enzyme cofactors: NAD+, NADH, NADP+, NADPH, thiamine pyrophosphate, pyridoxal 5′-phosphate, biotin, S-(5′-adenosyl)-l-methionine, coenzyme A (CoA), flavin mononucleotide (FMN), flavin adenine dinucleotide, acetyl-CoA, ATP, AMP, GTP, GDP, GMP, and CMP. In addition to the above components, 200 μm each of methionine sulfone, 3-aminopyrrolidine, MES, and trimesic acid were added to the reaction mixture as internal standards for CE-TOFMS analysis. Following the addition of 1 μg of purified protein to 100 μl of the above assay solution, the mixture was incubated for 1 h or longer at 37 °C. The reaction mixture was ultrafiltrated through a Millipore centrifugal membrane with 10,000 nominal molecular weight limit to remove the enzyme and stop the reaction. The filtrate was analyzed directly by CE-TOFMS in both positive and negative modes to profile cationic and anionic molecules, respectively. CE-TOFMS was carried out using an Agilent CE Capillary Electrophoresis System equipped with an Agilent 6210 Time-of-flight mass spectrometer, Agilent 1100 isocratic HPLC pump, Agilent G1603A CE-MS adapter kit, and Agilent G1607A CE-ESI-MS sprayer kit (Agilent Technologies, Waldbronn, Germany). The system was controlled by Agilent G2201AA ChemStation software for CE. Data acquisition was performed by Analyst QS Build: 7222 software for Agilent TOF (Applied Biosystems and MDS Sciex, Ontario, Canada). Instrumental conditions for separations and detections of metabolites were as follows. The cationic metabolites were separated on a fused silica capillary (50 μm × 100 cm) using 1 m formic acid as the electrolyte. The applied voltage was set at +30 kV. A solution of 50% (v/v) methanol/water was delivered as the sheath liquid at 10 μl/min (20Soga T. Heiger D.N. Anal. Chem. 2000; 72: 1236-1241Crossref PubMed Scopus (443) Google Scholar, 21Soga T. Ohashi Y. Ueno Y. Naraoka H. Tomita M. Nishioka T. J. Proteome Res. 2003; 2: 488-494Crossref PubMed Scopus (797) Google Scholar). Separations of anionic metabolites were carried out on a cationic polymer-coated SMILE (+) capillary (Nacalai Tesque, Kyoto, Japan) using 50 mm ammonium acetate (pH 8.5) as the electrolyte. The applied voltage was set at −30 kV. A solution of 5 mm ammonium acetate in 50% (v/v) methanol/water was delivered as the sheath liquid (21Soga T. Ohashi Y. Ueno Y. Naraoka H. Tomita M. Nishioka T. J. Proteome Res. 2003; 2: 488-494Crossref PubMed Scopus (797) Google Scholar, 22Soga T. Ueno Y. Naraoka H. Ohashi Y. Tomita M. Nishioka T. Anal. Chem. 2002; 74: 2233-2239Crossref PubMed Scopus (400) Google Scholar). For nucleotides, separations were performed using a fused silica capillary and 50 mm ammonium acetate (pH 7.5) as the electrolyte. The capillary was pretreated for 20 min with 25 mm ammonium acetate, 75 mm sodium phosphate buffer (pH 7.5). The applied voltage was set at −30 kV. A solution of 50% (v/v) methanol/water was delivered as the sheath liquid. Pressure (50 mbar) was applied to the capillary inlet during the run (23Soga T. Ishikawa T. Igarashi S. Sugawara K. Kakazu Y. Tomita M. J. Chromatogr. A. 2007; 1159: 125-133Crossref PubMed Scopus (79) Google Scholar). Electrospray ionization-TOFMS (ESI-TOFMS) was conducted in the positive ion mode (4000 V) for cationic metabolites, and the negative ion mode (3500 V) for anionic metabolites and nucleotides. Dry nitrogen gas was maintained at 10 p.s.i. Exact mass data were acquired over a 50–1000 m/z range (24Soga T. Baran R. Suematsu M. Ueno Y. Ikeda S. Sakurakawa T. Kakazu Y. Ishikawa T. Robert M. Nishioka T. Tomita M. J. Biol. Chem. 2006; 281: 16768-16776Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 25Ohashi Y. Hirayama A. Ishikawa T. Nakamura S. Shimizu K. Ueno Y. Tomita M. Soga T. Mol. Biosyst. 2008; 4: 135-147Crossref PubMed Scopus (205) Google Scholar). Data analysis was performed using a differential visualization tool that can highlight differences in the metabolite composition of two or more complex samples (26Baran R. Kochi H. Saito N. Suematsu M. Soga T. Nishioka T. Robert M. Tomita M. BMC Bioinformatics. 2006; 7: 530Crossref PubMed Scopus (138) Google Scholar). In some cases, metabolite quantification was performed using in-house software that detects peak features, performs migration time alignment, and peak area integration. Absolute quantification was performed using metabolite standards for calibration, when available. The acquisition of MS/MS spectra was performed using a Q-star XL (Applied Biosystems) instrument. Most of the conditions were identical to those in anionic metabolite analysis using CE-TOFMS. ESI-Q-TOFMS was conducted in the negative product ion scan mode; the ion spray voltage was set at −4000 V. Dry air (GS1) was maintained at 30 p.s.i. The declustering potentials 1 and 2, and the collision energy voltage were set at −30, −15, and −15, or −10 V, respectively. E. coli K12 BW25113 (laboratory stock) was used as parental strain in this study. The empty plasmid pCA24N and the plasmid containing an insert encoding His-tagged YihU (JW3853) were obtained from the ASKA library (18Kitagawa M. Ara T. Arifuzzaman M. Ioka-Nakamichi T. Inamoto E. Toyonaga H. Mori H. DNA Res. 2005; 12: 291-299Crossref PubMed Scopus (1036) Google Scholar). BW25113 was transformed with plasmid pCA24N or the yihU-containing plasmid, and the transformants were used for the experiment. The yihU knock-out strain was obtained from the Keio collection (27Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Mol. Syst. Biol. 2006; 2 (2006.0008)Crossref Scopus (5318) Google Scholar). E. coli was pre-cultured until mid-exponential phase in LB medium and an aliquot was inoculated into fresh LB followed by incubation at 37 °C with shaking. When the cells reached the mid-exponential phase (OD590 = 0.6), 0.5 mm isopropyl β-d-thiogalactopyranoside was added to induce yihU expression, and the culture was incubated at 37 °C until the stationary phase. At that point, 1-ml aliquots of the culture were incubated at 37 °C with or without 5 mm SSA (Sigma) for the indicated time period after which cells were collected to extract metabolites. For the experiments measuring resistance against SSA, cells were grown in LB medium at 37 °C to the mid to late exponential phase (OD590 = 1.0), then 1-ml aliquots of culture were treated with different concentrations of SSA and incubated for 1 h at room temperature. Cells were then washed twice with LB, and then spread onto LB plates to measure viable cells. Samples for intracellular metabolite measurements were processed as described previously with the following modifications (21Soga T. Ohashi Y. Ueno Y. Naraoka H. Tomita M. Nishioka T. J. Proteome Res. 2003; 2: 488-494Crossref PubMed Scopus (797) Google Scholar, 25Ohashi Y. Hirayama A. Ishikawa T. Nakamura S. Shimizu K. Ueno Y. Tomita M. Soga T. Mol. Biosyst. 2008; 4: 135-147Crossref PubMed Scopus (205) Google Scholar). Briefly, the culture (OD590 × sampling volume (ml) of culture = 5) was filtrated under vacuum using a 0.4-μm pore size filter. Cells on the membrane filter were immediately washed with Milli-Q water to remove extracellular components, and then quickly immersed in 2 ml of methanol containing 2.5 μm each of the internal standards, methionine sulfone, MES, and d-camphor 10-sulfonic acid. Dishes containing filters were sonicated for 30 s to resuspend the cells. A 1.6-ml portion of the cell suspension was transferred to a tube, and mixed with an equal volume of chloroform and 640 μl of Milli-Q water. After vortexing and centrifugation, the aqueous layer was recovered and ultrafiltrated by centrifugation at 9100 × g using Amicon Ultrafree-MC ultrafilter devices (Millipore Co.). The filtrate was dried, and then dissolved in 25 μl of Milli-Q water before CE-TOFMS analysis. As reported previously (17Saito N. Robert M. Kitamura S. Baran R. Soga T. Mori H. Nishioka T. Tomita M. J. Proteome Res. 2006; 5: 1979-1987Crossref PubMed Scopus (68) Google Scholar), we have been screening multiple uncharacterized enzyme-like proteins using generic assays based on metabolite profiling to discover novel activities. The activity of one such candidate, the putative E. coli dehydrogenase YihU, was assayed using a complex metabolite mixture prepared from yeast extract supplemented with defined cofactors, in the presence or absence of YihU protein as described under "Experimental Procedures." Following incubation, metabolites in the reaction mixture were profiled by CE-TOFMS. In this manner, we looked for metabolite level changes induced by YihU protein addition that can suggest the presence of enzymatic activity and reveal its substrates and products. Fig. 1 shows the anionic metabolite profiles obtained by CE-TOFMS analysis after in vitro reaction in the presence or absence of YihU protein. The levels of two anionic compounds (m/z 101.027 and 115.006) that were not prominent in the control reaction were found to significantly increase following incubation with YihU protein. These compounds were therefore candidates as products of the YihU enzymatic reaction. Increased levels of the anionic compounds were detected only in the presence of NAD+ or NADH, indicating that the reaction was NAD+/NADH-dependent and thus likely an oxidoreductase activity. There were no other significant changes in metabolite profiles of both cationic and anionic compounds beside noise-related false-positive signals (Fig. 1 and supplemental Fig. S1). In addition, there was no clear corresponding decrease in metabolite peaks that could have allowed to directly identify the potential substrate(s). We next attempted to identify the unknown anions (m/z 101.027 and 115.006) by comparing their accurate mass with the theoretical mass of compounds in the KEGG LIGAND data base (28Goto S. Okuno Y. Hattori M. Nishioka T. Kanehisa M. Nucleic Acids Res. 2002; 30: 402-404Crossref PubMed Scopus (253) Google Scholar). Among the resulting candidate compounds, those that were commercially available were analyzed by CE-TOFMS and CE-Q-TOFMS, and compared with the unknowns. The m/z ratio of one anion (m/z 101.027) was identical with that of SSA (within 1 ppm), whereas that of the other anion with m/z 115.006 corresponded with fumarate. There was perfect correspondence of CE migration times between the MERMAID reaction-produced unknowns and authentic standards (SSA and fumarate) when spiked into the MERMAID sample (supplemental Fig. S2, A and B). In addition, when the MS/MS spectrum of one of the anions (m/z 115) was compared with that of fumarate, the two showed clear similarities with two major product ions of m/z 59 and 71 (supplemental Fig. S2C). MS/MS spectral data for the m/z 101 anion could not be obtained due to its low abundance in the reaction mixture (<10 μm). However, both accurate mass and relative CE migration time (and MS/MS spectrum for the m/z 115 anion) strongly support the fact that SSA and fumarate were the two products specifically produced by the reaction catalyzed by YihU during MERMAID screening. To further investigate the enzymatic activity of YihU protein and because no clear substrate candidates were found during screening, we used a defined reaction mixture composed of only the pairs of identified products (SSA-NADH or NAD+, fumarate-NADH or NAD+) as substrates for the reverse reaction with YihU. When SSA and NADH were used for the reaction, the NADH peak disappeared with a concomitant appearance of an anionic peak of m/z 103.041 and NAD+ (Fig. 2A), indicating a NADH-dependent reduction of SSA by YihU. Although SSA must have been consumed in the reaction, its corresponding peak intensity only marginally decreased, probably due to saturation of the mass detector signal at the high concentration (5 mm) of SSA used. When a similar reaction was performed with fumarate and NADH or NAD+, no clear changes in metabolite levels were observed. We also tested succinate as substrate because fumarate might be generated by NAD+-dependent oxidation of succinate, but no enzymatic reaction was observed under these conditions. This result suggests that in vitro production of fumarate requires other unknown component(s) or cofactor(s) that were present in the original metabolite mixture of yeast extract but remained undetectable by CE-TOFMS (either neutral compounds or compounds below the detection limit). According to the BRENDA enzyme database (29Schomburg I. Chang A. Schomburg D. Nucleic Acids Res. 2002; 30: 47-49Crossref PubMed Scopus (340) Google Scholar), the NADH-dependent reduction of SSA by γ-hydroxybutyrate dehydrogenase (GHBDH; EC 1.1.1.61) generates GHB (Fig. 2B), a compound whose theoretical m/z value of 103.040 is in good agreement with the accurate mass of the unknown anion (m/z 103.041) measured by CE-TOFMS. We compared the profiles of the unknown anion with the GHB standard by CE-TOFMS and CE-Q-TOFMS analysis. There was perfect correspondence of CE migration time between the unknown anion and GHB when the latter was spiked into samples. The measured mass was within 1.1 ppm of that of GHB and the MS/MS spectrum of the unknown anion was nearly identical with that of GHB standard (Fig. 3). Moreover, the MS/MS fragmentation patterns of the GHB structural isomers, 2- and 3-hydroxybutyrate, were distinct from that of the unknown compound (supplemental Fig. S3). These results provide strong evidence that the unknown peak of m/z 103.041 is indeed GHB and therefore YihU is a novel E. coli GHBDH that can produce GHB from SSA. Sequence analysis by BLAST (blastp) (30Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (69731) Google Scholar) using the full-length amino acid sequence of YihU as query showed that the sequence is similar to the structurally conserved β-hydroxyisobutyrate and 6-phosphogluconate dehydrogenases family of enzymes. The highest scoring sequences are uncharacterized proteins from closely related organisms, Shigella flexneri (100% identity), Salmonella sp. (85% similarity), other bacteria, and E. coli strains, which are most likely YihU orthologs. InterProScan (31Quevillon E. Silventoinen V. Pillai S. Harte N. Mulder N. Apweiler R. Lopez R. Nucleic Acids Res. 2005; 33: W116-W120Crossref PubMed Scopus (1925) Google Scholar) results with the YihU sequence showed that the protein comprises the characteristic NAD(P)-binding Rossmann-like domain of 6-phosphogluconate dehydrogenase (PF03446). Including YihU, E. coli K12 (W3110) contains a total of four proteins annotated as members of the β-hydroxyisobutyrate dehydrogenase family (GenoBase). Fig. 4 shows the alignment of YihU with the three E. coli β-hydroxyisobutyrate dehydrogenase paralogs (GarR, GlxR, and YgbJ) and GHBDH from A. thaliana. As can be seen, amino acid similarity between YihU and other proteins was found throughout the length of the proteins with especially well conserved stretches in the previously reported dinucleotide cofactor binding, substrate binding, and catalytic domains (5Hawes J.W. Harper E.T. Crabb D.W. Harris R.A. FEBS Lett. 1996; 389: 263-267Crossref PubMed Scopus (37) Google Scholar, 6Njau R.K. Herndon C.A. Hawes J.W. Chem. Biol. Interact. 2001; 130–132: 785-791Crossref PubMed Scopus (26) Google Scholar) (Fig. 4). To further characterize YihU, its quaternary structure was analyzed by size exclusion chromatography. The protein eluted with an apparent native size of ∼143,000 (Kav value of 0.27) (supplemental Fig. S4). The monomeric molecular weight of YihU is predicted to be 31,154 as calculated from its amino acid sequence suggesting that the protein associates into a homotetramer, a common feature that was also observed for other β-hydroxyacid dehydrogenases in E. coli and Haemophilus influenzae (8Njau R.K. Herndon C.A. Hawes J.W. J. Biol. Chem. 2000; 275: 38780-38786Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The results of the original screening and subsequent confirmations suggested that YihU can catalyze the NADH-dependent reduction of SSA to GHB. To determine the substrate specificity of YihU and compare it with other members of the family, its enzymatic activity toward various metabolites was anal
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