RocG

• Description: trigger enzyme: catabolic glutamate dehydrogenase induced by arginine, ornithine or proline, subject to carbon catabolite repression
  
  

Gene name	rocG
Synonyms
Essential	no
Product	trigger enzyme: glutamate dehydrogenase (major)
Function	arginine utilization, controls the activity of GltC
Gene expression levels in SubtiExpress: rocG
Interactions involving this protein in SubtInteract: RocG
Metabolic function and regulation of this protein in SubtiPathways: rocG
MW, pI	46.2 kDa, 6.28
Gene length, protein length	1272 bp, 424 amino acids
Immediate neighbours	rocA, sivA
Sequences	Protein DNA DNA_with_flanks
Genetic context This image was kindly provided by SubtiList
Expression at a glance   PubMed

Categories containing this gene/protein

utilization of amino acids, glutamate metabolism, transcription factors and their control, trigger enzyme

This gene is a member of the following regulons

AbrB regulon, AhrC regulon, CcpA regulon, RocR regulon, SigL regulon

The gene

Basic information

• Locus tag: BSU37790

Phenotypes of a mutant

• Poor growth on complex media such as SP (sporulation medium). No growth in minimal media with arginine as the only carbon source. Rapid accumulation of suppressor mutants (gudB1)
• sensitive to ß-lactam antibiotics such as cefuroxime and to fosfomycin (suppressed by activation of gudB) due to the downregulation of the SigW regulon PubMed
• transcription profile of a rocG gudB mutant strain: GEO PubMed

Database entries

• BsubCyc: BSU37790

• DBTBS entry: [1]

• SubtiList entry: [2]

• GEO entry: [3] (rocG gudB mutant vs. wild type)

The protein

Basic information/ Evolution

• Catalyzed reaction/ biological activity: L-glutamate + H₂O + NAD⁺ = 2-oxoglutarate + NH₃ + NADH + H⁺ (according to Swiss-Prot), controls the activity of the GltC transcription activator PubMed

• Protein family: Glu/Leu/Phe/Val dehydrogenases family (according to Swiss-Prot)

• Paralogous protein(s): GudB

Extended information on the protein

• Kinetic information: K_M [glutamate] = 2.9 mM, K_M [ammonium] = 18 mM PubMed

• Domains:

• Modification:

• Cofactor(s): NAD⁺/NADH + H⁺

• Effectors of protein activity:

• Interactions:
  • RocG-GltC, this interaction prevents transcription activation of the gltA-gltB operon by GltC PubMed

• Localization:

Database entries

• BsubCyc: BSU37790

• Structure: 3K92 (super-repressor mutant that is capable of constitutive inactivation of GltC, E93K mutation) PubMed

• UniProt: P39633

• KEGG entry: [4]

• E.C. number: 1.4.1.2

Expression and regulation

• Operon: rocG PubMed

• Expression browser: rocG PubMed

• Sigma factor: SigL PubMed

• Regulation:
  • induced by arginine (RocR, AhrC), ornithine or proline PubMed
  • subject to carbon catabolite repression (CcpA) PubMed

• Regulatory mechanism:
  • RocR: transcription activation PubMed
  • AhrC: transcription activation PubMed
  • CcpA: transcription repression PubMed
  • AbrB: transcription repression PubMed

• Additional information:

Activation by RocR requires binding of RocR to a downstream element PubMed

• • number of protein molecules per cell (complex medium with amino acids, without glucose): 16024 PubMed

Biological materials

• Mutant: GP747 (spc), GP726 (aphA3), GP810 (del tet), GP1157 (cat) all available in Jörg Stülke's lab

• Expression vector:
  • expression of native rocG in B. subtilis: pGP529 (in pBQ200), available in Jörg Stülke's lab PubMed
  • for purification of RocG from E. coli carrying an N-terminal Strep-tag: pGP902 (in pGP172), a series of rocG variants is also available in pGP172, available in Jörg Stülke's lab
  • for expression/ purification from E. coli with N-terminal His-tag and thrombin cleavage site, in pWH844: pGP860, available in Jörg Stülke's lab
  • purification from B. subtilis with an N-terminal Strep-tag, for SPINE, (in pGP380): pGP1709, available in Jörg Stülke's lab

• lacZ fusion:

• GFP fusion:

• two-hybrid system: B. pertussis adenylate cyclase-based bacterial two hybrid system (BACTH), available in Jörg Stülke's lab

• Antibody: available in Jörg Stülke's lab

Labs working on this gene/protein

Linc Sonenshein, Tufts University, Boston, MA, USA Homepage

Jörg Stülke, University of Göttingen, Germany Homepage

Fabian Commichau University of Göttingen, Germany Homepage

References

Reviews

Katrin Gunka, Fabian M Commichau
Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation.
Mol Microbiol: 2012, 85(2);213-24
[PubMed:22625175] [WorldCat.org] [DOI] (I p)

Jason R Treberg, Margaret E Brosnan, Malcolm Watford, John T Brosnan
On the reversibility of glutamate dehydrogenase and the source of hyperammonemia in the hyperinsulinism/hyperammonemia syndrome.
Adv Enzyme Regul: 2010, 50(1);34-43
[PubMed:19895831] [WorldCat.org] [DOI] (I p)

Victoria I Bunik, Alisdair R Fernie
Metabolic control exerted by the 2-oxoglutarate dehydrogenase reaction: a cross-kingdom comparison of the crossroad between energy production and nitrogen assimilation.
Biochem J: 2009, 422(3);405-21
[PubMed:19698086] [WorldCat.org] [DOI] (I e)

N M Brunhuber, J S Blanchard
The biochemistry and enzymology of amino acid dehydrogenases.
Crit Rev Biochem Mol Biol: 1994, 29(6);415-67
[PubMed:7705101] [WorldCat.org] [DOI] (P p)

R C Hudson, R M Daniel
L-glutamate dehydrogenases: distribution, properties and mechanism.
Comp Biochem Physiol B: 1993, 106(4);767-92
[PubMed:8299344] [WorldCat.org] [DOI] (P p)

Enzymatic activity of RocG

Lorena Stannek, Martin J Thiele, Till Ischebeck, Katrin Gunka, Elke Hammer, Uwe Völker, Fabian M Commichau
Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.
Environ Microbiol: 2015, 17(9);3379-90
[PubMed:25711804] [WorldCat.org] [DOI] (I p)

Kenji Manabe, Yasushi Kageyama, Takuya Morimoto, Tadahiro Ozawa, Kazuhisa Sawada, Keiji Endo, Masatoshi Tohata, Katsutoshi Ara, Katsuya Ozaki, Naotake Ogasawara
Combined effect of improved cell yield and increased specific productivity enhances recombinant enzyme production in genome-reduced Bacillus subtilis strain MGB874.
Appl Environ Microbiol: 2011, 77(23);8370-81
[PubMed:21965396] [WorldCat.org] [DOI] (I p)

Katrin Gunka, Joseph A Newman, Fabian M Commichau, Christina Herzberg, Cecilia Rodrigues, Lorraine Hewitt, Richard J Lewis, Jörg Stülke
Functional dissection of a trigger enzyme: mutations of the bacillus subtilis glutamate dehydrogenase RocG that affect differentially its catalytic activity and regulatory properties.
J Mol Biol: 2010, 400(4);815-27
[PubMed:20630473] [WorldCat.org] [DOI] (I p)

Shigeki Kada, Masahiro Yabusaki, Takayuki Kaga, Hitoshi Ashida, Ken-ichi Yoshida
Identification of two major ammonia-releasing reactions involved in secondary natto fermentation.
Biosci Biotechnol Biochem: 2008, 72(7);1869-76
[PubMed:18603778] [WorldCat.org] [DOI] (I p)

Fabian M Commichau, Katrin Gunka, Jens J Landmann, Jörg Stülke
Glutamate metabolism in Bacillus subtilis: gene expression and enzyme activities evolved to avoid futile cycles and to allow rapid responses to perturbations of the system.
J Bacteriol: 2008, 190(10);3557-64
[PubMed:18326565] [WorldCat.org] [DOI] (I p)

Md Iqbal Hassan Khan, Kousuke Ito, Hyeung Kim, Hiroyuki Ashida, Takahiro Ishikawa, Hitoshi Shibata, Yoshihiro Sawa
Molecular properties and enhancement of thermostability by random mutagenesis of glutamate dehydrogenase from Bacillus subtilis.
Biosci Biotechnol Biochem: 2005, 69(10);1861-70
[PubMed:16244435] [WorldCat.org] [DOI] (P p)

Iqbal Hassan Khan, Hyeung Kim, Hiroyuki Ashida, Takahiro Ishikawa, Hitoshi Shibata, Yoshihiro Sawa
Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis.
Biosci Biotechnol Biochem: 2005, 69(9);1802-5
[PubMed:16195607] [WorldCat.org] [DOI] (P p)

B R Belitsky, A L Sonenshein
Role and regulation of Bacillus subtilis glutamate dehydrogenase genes.
J Bacteriol: 1998, 180(23);6298-305
[PubMed:9829940] [WorldCat.org] [DOI] (P p)

Additional publications

Lorena Stannek, Richard Egelkamp, Katrin Gunka, Fabian M Commichau
Monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains.
J Vis Exp: 2014, (83);e51196
[PubMed:24473333] [WorldCat.org] [DOI] (I e)

Kenji Manabe, Yasushi Kageyama, Takuya Morimoto, Eri Shimizu, Hiroki Takahashi, Shigehiko Kanaya, Katsutoshi Ara, Katsuya Ozaki, Naotake Ogasawara
Improved production of secreted heterologous enzyme in Bacillus subtilis strain MGB874 via modification of glutamate metabolism and growth conditions.
Microb Cell Fact: 2013, 12;18
[PubMed:23419162] [WorldCat.org] [DOI] (I e)

Li-Li Chen, Jia-Le Wang, Yu Hu, Bing-Jun Qian, Xiao-Min Yao, Jing-Fang Wang, Jian-Hua Zhang
Computational design of glutamate dehydrogenase in Bacillus subtilis natto.
J Mol Model: 2013, 19(4);1919-27
[PubMed:23338837] [WorldCat.org] [DOI] (I p)

Yong Heon Lee, Anthony W Kingston, John D Helmann
Glutamate dehydrogenase affects resistance to cell wall antibiotics in Bacillus subtilis.
J Bacteriol: 2012, 194(5);993-1001
[PubMed:22178969] [WorldCat.org] [DOI] (I p)