Corynebacterial Protein Kinase G Controls 2-Oxoglutarate Dehydrogenase Activity via the Phosphorylation Status of the OdhI Protein
2006; Elsevier BV; Volume: 281; Issue: 18 Linguagem: Inglês
10.1074/jbc.m512515200
ISSN1083-351X
AutoresAxel Niebisch, Armin Kabus, Christian Schultz, Brita Weil, Michael Bott,
Tópico(s)Enzyme Structure and Function
ResumoA novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the ΔpknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ΔodhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a Ki value of 2.4 nm. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle. A novel regulatory mechanism for control of the ubiquitous 2-oxoglutarate dehydrogenase complex (ODH), a key enzyme of the tricarboxylic acid cycle, was discovered in the actinomycete Corynebacterium glutamicum, a close relative of important human pathogens like Corynebacterium diphtheriae and Mycobacterium tuberculosis. Based on the finding that a C. glutamicum mutant lacking serine/threonine protein kinase G (PknG) was impaired in glutamine utilization, proteome comparisons led to the identification of OdhI as a putative substrate of PknG. OdhI is a 15-kDa protein with a forkhead-associated domain and a homolog of mycobacterial GarA. By using purified proteins, PknG was shown to phosphorylate OdhI at threonine 14. The glutamine utilization defect of the ΔpknG mutant could be abolished by the additional deletion of odhI, whereas transformation of a ΔodhI mutant with a plasmid encoding OdhI-T14A caused a defect in glutamine utilization. Affinity purification of OdhI-T14A led to the specific copurification of OdhA, the E1 subunit of ODH. Because ODH is essential for glutamine utilization, we assumed that unphosphorylated OdhI inhibits ODH activity. In fact, OdhI was shown to strongly inhibit ODH activity with a Ki value of 2.4 nm. The regulatory mechanism described offers a molecular clue for the reduced ODH activity that is essential for the industrial production of 1.5 million tons/year of glutamate with C. glutamicum. Moreover, because this signaling cascade is likely to operate also in mycobacteria, our results suggest that the attenuated pathogenicity of mycobacteria lacking PknG might be caused by a disturbed tricarboxylic acid cycle. Increasing numbers of eukaryotic-like serine/threonine protein kinases found in bacteria implicate that they play important roles in cell signaling, but their targets and specific functions are largely unknown (1Kennelly P.J. FEMS Microbiol. Lett. 2002; 206: 1-8Crossref PubMed Google Scholar). The genome of the important human pathogen Mycobacterium tuberculosis encodes 11 members of this protein family (2Av-Gay Y. Everett M. Trends Microbiol. 2000; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Among these, protein kinase G (PknG) 2The abbreviations used are: PknG, protein kinase G; FHA, forkhead-associated; ODH, 2-oxoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. 2The abbreviations used are: PknG, protein kinase G; FHA, forkhead-associated; ODH, 2-oxoglutarate dehydrogenase; PDH, pyruvate dehydrogenase; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. gained particular interest because it was reported to inhibit phagosome-lysosome fusion, thus allowing for intracellular survival of mycobacteria. Deletion of the pknG gene in Mycobacterium bovis BCG resulted in lysosomal localization and mycobacterial cell death in infected macrophages. PknG was detected in the cytosol of infected macrophages and was therefore suggested to interfere with host cell signaling pathways (3Walburger A. Koul A. Ferrari G. Nguyen L. Prescianotto-Baschong C. Huygen K. Klebl B. Thompson C. Bacher G. Pieters J. Science. 2004; 304: 1800-1804Crossref PubMed Scopus (439) Google Scholar). A pknG deletion mutant of M. tuberculosis displayed decreased viability upon infection of immunocompetent mice but also reduced growth in vitro (4Cowley S. Ko M. Pick N. Chow R. Downing K.J. Gordhan B.G. Betts J.C. Mizrahi V. Smith D.A. Stokes R.W. Av-Gay Y. Mol. Microbiol. 2004; 52: 1691-1702Crossref PubMed Scopus (193) Google Scholar), implying that PknG function is not restricted to the pathogenic life style. This is supported by the fact that genes encoding PknG homologs are not only present in pathogenic mycobacteria but also in all other members of the suborder Corynebacterineae with known genome sequence, i.e. species of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus, as well as in Streptomyces species. To determine the function of PknG, we chose Corynebacterium glutamicum, a nonpathogenic species used for biotechnological amino acid production (5Eggeling L. Bott M. Handbook of Corynebacterium glutamicum. CRC Press, Inc., Boca Raton, FL2005Crossref Google Scholar), which already proved useful for understanding the function of homologous genes in M. tuberculosis (6Alderwick L.J. Radmacher E. Seidel M. Gande R. Hitchen P.G. Morris H.R. Dell A. Sahm H. Eggeling L. Besra G.S. J. Biol. Chem. 2005; 280: 32362-32371Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar).EXPERIMENTAL PROCEDURESBacterial Strains and Culture Conditions—The strains and plasmids used in this study are listed in Table 1. C. glutamicum strains were cultivated aerobically in shake flasks at 150 rpm and 30 °C in brain heart infusion medium (Difco) or CGXII minimal medium (7Keilhauer C. Eggeling L. Sahm H. J. Bacteriol. 1993; 175: 5595-5603Crossref PubMed Google Scholar) with 200 mm glucose. For growth experiments on glutamine as carbon and nitrogen source, C. glutamicum was precultured overnight in brain heart infusion medium with 100 mm glucose. Cells were harvested, washed with 0.9% NaCl, and used to inoculate the main cultures in glutamine medium (modified CGXII lacking ammonium sulfate and urea and containing 100 mm glutamine) supplemented with 5 mm glucose. Samples for determination of intracellular metabolites and for proteome analysis were taken after 12 h of cultivation when the ΔpknG strain just became stationary after glucose depletion. For the determination of growth rates, cells grown in brain heart infusion medium with glucose were cultivated in glutamine medium with 20 mm glucose. The cells from this second preculture were used to inoculate glutamine medium without glucose. For all cloning purposes, Escherichia coli DH5α was used and routinely grown in Luria-Bertani medium at 37 °C. When appropriate, kanamycin was added at concentrations of 25 mg/liter (C. glutamicum) or 50 mg/liter (E. coli).TABLE 1Plasmids and C. glutamicum strains used in this studyDescriptionRef.StrainsATCC13032Biotin-auxotrophic wild-type strain39Kinoshita S. Udaka S. Shimono M. J. Gen. Appl. Microbiol. 1957; 3: 193-205Crossref Scopus (413) Google ScholarΔpknGWild type derivative with in-frame deletion of pknG (cg3046)This workΔglnHWild type derivative with in-frame deletion of glnH (cg3045)This workΔglnXWild type derivative with in-frame deletion of glnX (cg3044)This workΔodhIWild type derivative with in-frame deletion of odhI (cg1630)This workΔpknGΔodhIΔpknG derivative with additional in-frame deletion of odhIThis workWT-odhAStWild type derivative with plasmid pK18mob-odhASt integrated into the chromosomal odhA gene (cg1280) adding a Strep-tag-II coding sequence before the odhA stop codonThis workΔpknG-odhAStAs above but ΔpknG derivativeThis workWT-aceEStWild type derivative with plasmid pK18mob-aceESt integrated into the chromosomal aceE gene (cg2466) adding a Strep-tag-II coding sequence before the aceE stop codonThis workΔpknG-aceEStAs above but ΔpknG derivativeThis workPlasmidspEKEx2KanR, C. glutamicum expression vector for IPTG-inducible gene expression40Eikmanns B.J. Kleinertz E. Liebl W. Sahm H. Gene (Amst.). 1991; 102: 93-98Crossref PubMed Scopus (187) Google ScholarpEKEx2-pknGStpEKEx2 derivative containing the C. glutamicum pknG gene with the native ribosome-binding site and a Strep-tag-II coding sequence before the pknG stop codonThis workpAN3KKanR, derivative of the E. coli expression vector pASK-IBA3C (IBA, Göttingen, Germany) for anhydrotetracycline-inducible production of C-terminally Strep-tagged proteins, contains the KanR gene and the C. glutamicum replicon from pJC1 allowing plasmid replication and gene expression in C. glutamicumThis workpAN3K-odhI and derivativespAN3K derivative containing the C. glutamicum wild-type odhI gene (obtained by PCR with primers OdhI-for-1/rev-1) or mutated odhI genes with Thr-14 or -15 codons exchanged to alanine codonsThis workpJC1KanR, E. coli-C. glutamicum shuttle vector41Cremer J. Eggeling L. Sahm H. Mol. Gen. Genet. 1990; 220: 478-480Crossref Scopus (71) Google ScholarpJC1-odhI and derivatives (T14A, T15A)pJC1 derivatives containing the C. glutamicum wild-type odhI gene (obtained by PCR with primers OdhI-for-2/-rev-2) with its native promoter or mutated odhI genes with Thr-14 or -15 codons exchanged to alanine codons; a Strep-tag-II coding sequence before the odhI stop codon was introduced by the reverse primerThis workpK18mobKanR, E. coli vector unable to replicate in C. glutamicum42Schäfer A. Tauch A. Jäger W. Kalinowski J. Thierbach G. Pühler A. Gene (Amst.). 1994; 145: 69-73Crossref PubMed Scopus (2124) Google ScholarpK18m-odhAStpK18mob derivative containing a 625-bp PCR product covering the 3′-terminal end of the odhA gene elongated with a Strep-tag-II coding sequence before the stop codonThis workpK18m-aceEStpK18mob derivative containing a 578-bp PCR product covering the 3′-terminal end of the aceE gene elongated with a Strep-tag-II coding sequence before the stop codonThis workpK19mobsacBKanR, E. coli vector for generating C. glutamicum deletion mutants42Schäfer A. Tauch A. Jäger W. Kalinowski J. Thierbach G. Pühler A. Gene (Amst.). 1994; 145: 69-73Crossref PubMed Scopus (2124) Google ScholarpK19ms-ΔpknGpK19mobsacB derivative containing a crossover PCR product (primer ΔpknG-1-4) that covers the flanking regions of the pknG geneThis workpK19ms-ΔglnHpK19mobsacB derivative containing a crossover PCR product (primer ΔglnH-1-4) that covers the flanking regions of the glnH geneThis workpK19ms-ΔglnXpK19mobsacB derivative containing a crossover PCR product (primer ΔglnX-1-4) that covers the flanking regions of the glnX geneThis workpK19ms-ΔodhIpK19mobsacB derivative containing a crossover PCR product (primer ΔodhI-1-4) that covers the flanking regions of the odhI geneThis work Open table in a new tab Construction of Plasmids and Mutants—The oligonucleotides used as PCR primer in this study are listed in Table S1. Plasmids were constructed by standard molecular genetic methods and confirmed by DNA sequence analysis. Defined C. glutamicum deletion mutants were constructed by crossover PCR and double homologous recombination using the suicide vector pK19mobsacB (8Niebisch A. Bott M. Arch. Microbiol. 2001; 175: 282-294Crossref PubMed Scopus (151) Google Scholar). All deletions were verified by Southern blot analysis. Site-directed mutagenesis of odhI was carried out by PCR using the mutagenic primer pairs T14A-for/-rev and T15A-for/-rev. For construction of C. glutamicum strains synthesizing Strep-tagged OdhA or Strep-tagged AceE, 3′-terminal fragments of the respective genes were amplified with the primer pairs odhA-for/odhA-rev and aceE-for/aceE-rev. The reverse primers introduced a Strep-tag-II coding sequence before the stop codon. After cloning the PCR products into the suicide vector pK18mob, the resulting plasmids were integrated into the C. glutamicum chromosome by a single homologous recombination event. Recombinant strains were selected on agar plates containing kanamycin.Determination of Internal Glutamine/Glutamate Concentrations—Culture samples containing 0.5–1 mg of biomass (dry weight) were rapidly filtered through glass fiber disks (Millipore), and the filter-bound cells were washed with 0.9% NaCl at room temperature (9Wittmann C. Krömer J.O. Kiefer P. Binz T. Heinzle E. Anal. Biochem. 2004; 327: 135-139Crossref PubMed Scopus (207) Google Scholar). Internal metabolites were extracted by incubating harvested cells in 1.3 ml of 50 μm ornithine for 15 min at 95 °C. Amino acids in the extracts were quantified by high pressure liquid chromatography after precolumn derivatization with o-phthaldialdehyde. Internal concentrations were calculated using a correlation of dry weight (mg/ml) = 0.25 × A600 and a cytoplasmic volume of 1.5 μl/mg dry weight (10Krämer R. Lambert C. Hoischen C. Ebbighausen H. Eur. J. Biochem. 1990; 194: 929-935Crossref PubMed Scopus (77) Google Scholar). The correlation between dry weight and OD600 was determined for three independent cultures of wild type and ΔpknG mutant and was found to be identical.Two-dimensional Gel Electrophoresis and Protein Identification—Cytosolic proteins were isolated and separated by two-dimensional gel electrophoresis as described (11Schaffer S. Weil B. Nguyen V.D. Dongmann G. Günther K. Nickolaus M. Hermann T. Bott M. Electrophoresis. 2001; 22: 4404-4422Crossref PubMed Scopus (123) Google Scholar). Protein identification from Coomassie-stained one- or two-dimensional gels and analysis of the OdhI phosphorylation site was performed by peptide mass fingerprinting of tryptic digests and post-source decay analysis of the phosphorylated peptide using a Voyager DE-STR mass spectrometer (Applied Biosystems, Weiterstadt, Germany) as described (11Schaffer S. Weil B. Nguyen V.D. Dongmann G. Günther K. Nickolaus M. Hermann T. Bott M. Electrophoresis. 2001; 22: 4404-4422Crossref PubMed Scopus (123) Google Scholar).Preparation of Cell-free Extracts and Protein Purification by Affinity Chromatography—All steps were performed at 4 °C. Generally, cells were resuspended in buffer A (100 mm Tris/HCl, pH 8.0, 150 mm NaCl) containing Complete EDTA-free protease inhibitor (Roche Diagnostics) and disrupted by sonication or French press treatment. Cell debris was removed by low speed centrifugation (18,000 × g for 10 min). Prior to purification, cell extracts from C. glutamicum were incubated with avidin (50 μg/mg protein) for 30 min to reduce copurification of the biotinylated proteins pyruvate carboxylase and acyl-CoA carboxylase. Strep-tagged PknG, OdhI, and mutated derivatives were purified from C. glutamicum ΔpknG/pEKEx2-pknGSt and E. coli DH5α containing pAN3K-odhI, pAN3K-odhI-T14A, or pAN3K-odhI-T15A, respectively, by affinity chromatography on Strep-Tactin-Sepharose columns (1-ml bed volume, IBA, Göttingen, Germany). Cell extracts (≤5 mg of protein/ml) were applied to the column and washed with 6 ml of buffer A, and bound proteins were eluted in 3 ml of buffer A with 2.5 mm desthiobiotin. Fractions containing the purified proteins were concentrated by ultrafiltration and stored frozen at –20 °C in elution buffer containing 20% (v/v) glycerol. For copurification attempts of OdhI and OdhA on analytical scale, Strep-Tactin-coated magnetic beads (IBA, Göttingen) were used. 10 mg of beads were activated according to the supplier's instructions, equilibrated in buffer A, and mixed with 2 ml of cell extract. After 1 h of incubation with occasional shaking, the beads were separated and washed five times with 200 μl of buffer A, and bound proteins were eluted in 100 μl of buffer A with 2.5 mm desthiobiotin.For 2-oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) activity assays and for purification of active ODH·PDH complexes, cells were disrupted in buffer B (50 mm TES/NaOH, pH 7.7, 10 mm MgCl2) with 30% (v/v) glycerol. Cell extracts for enzyme assays were gel-filtrated in the same buffer on PD10 columns (Amersham Biosciences) to remove interfering intracellular metabolites. ODH·PDH complexes were purified on Strep-Tactin-coated magnetic beads or Strep-Tactin-Sepharose (for subsequent enzyme assays) essentially as described above, but cell extracts were diluted with buffer B to 15% (v/v) glycerol, and buffer A was replaced by buffer B containing 10% (v/v) glycerol.Enzyme Assays—Autokinase activity of PknG and PknG-dependent phosphorylation of OdhI was assayed in kinase buffer (25 mm Tris/HCl, pH 7.5, 5 mm MgCl2, 2 mm MnCl2, 1 mm dithiothreitol) as described (12Koul A. Choidas A. Tyagi A.K. Drlica K. Singh Y. Ullrich A. Microbiology. 2001; 147: 2307-2314Crossref PubMed Scopus (90) Google Scholar). Oxoglutarate dehydrogenase and pyruvate dehydrogenase activity were measured at 30 °C in a photometric assay by following the initial increase in absorbance of NADH at 340 nm (13Shiio I. Ujigawatakeda K. Agric. Biol. Chem. 1980; 44: 1897-1904Crossref Scopus (6) Google Scholar). Assays containing buffer B with 3 mm l-cysteine, 0.9 mm thiamine diphosphate, 2 mm NAD+, 50 μm chlorpromazine (to prevent reoxidation of NADH (14Weinstein E.A. Yano T. Li L.S. Avarbock D. Avarbock A. Helm D. McColm A.A. Duncan K. Lonsdale J.T. Rubin H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4548-4553Crossref PubMed Scopus (222) Google Scholar)), 25–100 μl of cell extract, or purified ODH·PDH complexes and up to 30.4 nm OdhI were preincubated for 10 min at 30 °C before the reaction was started by addition of 1.5 mm 2-oxoglutarate or pyruvate and 0.2 mm coenzyme A. An activity of 1 unit refers to 1 μmol of NADH formed per min.Miscellaneous—Protein concentrations were determined with the Bradford protein assay using bovine serum albumin as standard. Glutamine uptake rates of cells grown in CGXII/glucose to the mid-log phase were determined using 1-[14C]glutamine and a filtration assay as described (15Siewe R.M. Weil B. Krämer R. Arch. Microbiol. 1995; 164: 98-103Crossref Scopus (16) Google Scholar).RESULTSPknG Is Required for Glutamine Utilization in C. glutamicum—The C. glutamicum pknG gene (cg3046) is located in a putative operon (Fig. 1A) with two genes, cg3044 (glnX) and cg3045 (glnH), that encode a hypothetical membrane protein with four potential transmembrane helices and a putative "periplasmic" glutamine-binding lipoprotein, respectively (16Kalinowski J. Bathe B. Bartels D. Bischoff N. Bott M. Burkovski A. Dusch N. Eggeling L. Eikmanns B.J. Gaigalat L. Goesmann A. Hartmann M. Huthmacher K. Krämer R. Linke B. McHardy A.C. Meyer F. Möckel B. Pfefferle W. Pühler A. Rey D.A. Rückert C. Rupp O. Sahm H. Wendisch V.F. Wiegrabe I. Tauch A. J. Biotechnol. 2003; 104: 5-25Crossref PubMed Scopus (754) Google Scholar). This gene arrangement is conserved in bacteria containing pknG homologs except for Streptomyces coelicolor and Streptomyces avermitilis where an additional open reading frame is present between glnH and pknG. Because GlnH homologs of other bacteria, e.g. E. coli or Bacillus subtilis, are part of ABC transporters for high affinity glutamine uptake, we tested the effect of an in-frame pknG deletion in C. glutamicum on glutamine utilization. As shown in Fig. 1B, C. glutamicum wild type grew on minimal medium agar plates with glutamine as sole carbon and nitrogen source, whereas growth of the ΔpknG mutant was severely inhibited. This phenotype could be complemented by plasmid-borne pknG (data not shown). In liquid glutamine medium, C. glutamicum wild type showed a growth rate of 0.17–0.20 h–1 and formed 5 g of cell dry weight liter–1 within 20 h. The ΔpknG strain showed no growth within this period. The mutant started to grow only after a prolonged incubation. This growth was probably because of suppressor mutations, because the lag phase of different cultures varied considerably, and cells from these cultures could subsequently grow immediately on glutamine agar plates. No significant growth differences were observed on rich medium or glucose minimal medium (data not shown). To study the role of GlnX and GlnH for glutamine utilization, in-frame deletion mutants of C. glutamicum lacking either glnX or glnH were constructed (Fig. 1A). On glutamine agar plates, both mutants showed a growth defect (Fig. 1B). In liquid glutamine medium, the ΔglnX mutant showed a similar phenotype as the ΔpknG mutant. There was no growth within 50 h, but upon prolonged incubation suppressor mutants started to grow. By contrast, the ΔglnH mutant was able to grow in liquid glutamine medium, with growth rates varying from 0.14 to 0.16 h–1. From these results, a function of PknG, GlnX, and GlnH in glutamine uptake or metabolism could be inferred.C. glutamicum possesses a secondary Na+-dependent glutamine uptake system that has not yet been genetically identified (15Siewe R.M. Weil B. Krämer R. Arch. Microbiol. 1995; 164: 98-103Crossref Scopus (16) Google Scholar). Transport assays were performed to study whether GlnH and GlnX are part of a glutamine transporter that is regulated by PknG. However, the C. glutamicum mutant strains lacking pknG, glnX, or glnH showed at least 80% of the glutamine transport activity of the wild type (data not shown), indicating that the defect of the mutants in glutamine utilization is not because of an impaired glutamine uptake. A similar result was reported for a M. bovis BCG ΔpknG mutant (17Nguyen L. Walburger A. Houben E. Koul A. Muller S. Morbitzer M. Klebl B. Ferrari G. Pieters J. J. Bacteriol. 2005; 187: 5852-5856Crossref PubMed Scopus (52) Google Scholar). Measurement of the internal amino acid concentrations revealed that the glutamate level was 2-fold higher in the ΔpknG mutant compared with the wild type and the complemented mutant, whereas the glutamine level was only slightly increased (Fig. 1C). Therefore, a defect in glutamate catabolism was likely to be responsible for the defect of the ΔpknG mutant in glutamine utilization.Identification of an in Vivo Substrate of PknG—To find the molecular basis for inhibition of glutamate catabolism by PknG, we searched for in vivo phosphorylation substrates of PknG by proteome analysis of C. glutamicum wild type, ΔpknG mutant, and complemented mutant grown on 100 mm glutamine and 5 mm glucose as carbon sources. Comparison of Coomassie-stained two-dimensional gels revealed a series of three spots in the acidic, low molecular mass range that clearly differed between the wild type and complemented ΔpknG strain on the one hand and the ΔpknG mutant on the other hand (Fig. 1D). All three spots were identified by mass spectrometry as the protein encoded by gene cg1630, which was designated OdhI. OdhI (143 amino acid residues, 15,402 Da) is a homolog (69% sequence identity) of Mycobacterium smegmatis GarA (18Belanger A.E. Hatfull G.F. J. Bacteriol. 1999; 181: 6670-6678Crossref PubMed Google Scholar) and contains a forkhead-associated (FHA) domain, which binds phosphothreonine epitopes on proteins and mediates phosphorylation-dependent protein-protein interactions (19Pallen M. Chuadhuri R. Khan A. Trends Microbiol. 2002; 10: 556-563Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The pI values of 4.64, 4.56, and 4.47 observed for the three OdhI spots were close to the theoretical values calculated for unphosphorylated, monophosphorylated, and doubly phosphorylated OdhI protein, respectively. The amount of the different OdhI isoforms was relatively quantified by densitometric analysis of the two-dimensional gels. In the wild type 61.5 ± 6.4% of total OdhI was present in the monophosphorylated and 5.5 ± 3.9% in the doubly phosphorylated form (all data are mean values ± S.D. of three gels from independent cultures). The complemented ΔpknG mutant showed no significant differences with corresponding values of 53.1 ± 14.3 and 2.0 ± 1.8%, respectively. In contrast, the ΔpknG mutant contained only 5.9 ± 0.1% monophosphorylated OdhI, and the doubly phosphorylated form was undetectable. These data suggest that OdhI is phosphorylated by PknG but also by at least one additional protein kinase. Besides PknG, C. glutamicum possesses homologs of the M. tuberculosis serine/threonine protein kinases PknA, PknB, and PknL (2Av-Gay Y. Everett M. Trends Microbiol. 2000; 8: 238-244Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), encoded by the genes cg0059, cg0057, and cg2388, respectively. As M. tuberculosis PknB was recently shown to phosphorylate mycobacterial GarA in vitro (20Villarino A. Duran R. Wehenkel A. Fernandez P. England P. Brodin P. Cole S.T. Zimny-Arndt U. Jungblut P.R. Cervenansky C. Alzari P.M. J. Mol. Biol. 2005; 350: 953-963Crossref PubMed Scopus (128) Google Scholar), PknB is a favorite candidate for PknG-independent OdhI phosphorylation.To confirm that OdhI is a substrate of PknG, both proteins were synthesized as Strep-tagged derivatives and purified to apparent homogeneity. As shown by in vitro kinase assays, PknG catalyzed autophosphorylation and transphosphorylation of OdhI, whereas OdhI alone did not incorporate 32P (Fig. 2A). By comparing the tryptic peptide mass fingerprints of phosphorylated and unphosphorylated OdhI, the amino-terminal peptide covering amino acid residues 2–19 was found to be the only one phosphorylated by PknG (Fig. 3, A and B). Post-source decay analysis of this peptide identified threonine 14 as the phosphorylated residue (Fig. 3C). This result was verified by in vitro kinase assays, which showed that a T14A exchange in OdhI abolished PknG-dependent phosphorylation (Fig. 2A). In contrast, a T15A exchange only reduced the phosphorylation efficiency, indicating that Thr-15 might be involved in the binding of OdhI to PknG. OdhI/GarA homologous proteins with a strictly conserved ETTS motif in the amino-terminal region occur in all species that contain PknG homologs (Fig. 2B). The first threonine residue of this motif is the one phosphorylated by PknG in C. glutamicum, and the second threonine residue corresponds to Thr-22 of GarA from M. tuberculosis that was shown to be phosphorylated by PknB (20Villarino A. Duran R. Wehenkel A. Fernandez P. England P. Brodin P. Cole S.T. Zimny-Arndt U. Jungblut P.R. Cervenansky C. Alzari P.M. J. Mol. Biol. 2005; 350: 953-963Crossref PubMed Scopus (128) Google Scholar).FIGURE 2Phosphorylation of OdhI by PknG and role of OdhI for glutamine utilization. A, autoradiogram showing in vitro phosphorylation of OdhI by PknG at threonine 14. A T14A exchange in OdhI abolishes phosphorylation, whereas a T15A exchange does not. 61 pmol of OdhI or the indicated variants were incubated with 5.6 pmol of PknG and 37 kBq [γ-32P]ATP for 30 min at 37 °C, denatured, and completely loaded on a 15% SDS gel. B, alignment of the amino-terminal regions of OdhI homologs from different Actinomycetales. The filled circle denotes the residue in C. glutamicum OdhI that is phosphorylated by PknG and the open circle the residue of the M. tuberculosis OdhI homolog (designated GarA), which is phosphorylated in vitro by PknB (20Villarino A. Duran R. Wehenkel A. Fernandez P. England P. Brodin P. Cole S.T. Zimny-Arndt U. Jungblut P.R. Cervenansky C. Alzari P.M. J. Mol. Biol. 2005; 350: 953-963Crossref PubMed Scopus (128) Google Scholar). Ce, Corynebacterium efficiens; Cj, Corynebacterium jeikeium; Mp, Mycobacterium avium subsp. paratuberculosis; Mb, M. bovis; Mt, M. tuberculosis; Ms, M. smegmatis; Rf, Rhodococcus fascians; Sa, S. avermitilis; Sc, S. coelicolor. C, growth with glutamine as sole carbon source of the following C. glutamicum strains: 1, wild type; 2, ΔpknG; 3, ΔodhI; 4, ΔpknGΔodhI; 5, ΔodhI/pJC1; 6, ΔodhI/pJC1-odhI-T14A; 7, ΔodhI/pJC1-odhI-T15A.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Mapping of the PknG-dependent phosphorylation site of OdhI. Peptide mass fingerprints of tryptic digests of unphosphorylated (A) and in vitro phosphorylated (B) Strep-tagged OdhI. Peaks are labeled with their monoisotopic masses and the assigned amino acid residues are shown in parentheses. The covered sequence (87% of 153 amino acids) included all serine and threonine residues. The putative phosphopeptide is marked with an asterisk. C, post-source decay analysis of the OdhI phosphopeptide (average mass 2059.0). For noise filtering and smoothing, the Data Explorer software (Applied Biosystems) was used. Fragment ions attributable to the y-series within a mass accuracy of 0.7 Da were labeled. β-Elimination of phosphoric acid (mass shift –98) is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Functional Characterization of the PknG Substrate OdhI—To address the in vivo function of OdhI, in particular its involvement in glutamine utilization, its structural gene was deleted in C. glutamicum wild type as well as in the ΔpknG mutant. Addit
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