scholarly journals In vitro reconstitution and characterization of pyruvate dehydrogenase and 2‐oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum

2020 ◽  
Vol 9 (10) ◽  
Author(s):  
Hirokazu Kinugawa ◽  
Naoko Kondo ◽  
Ayano Komine‐Abe ◽  
Takeo Tomita ◽  
Makoto Nishiyama ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Hybrid Complex ◽  
In Vitro Reconstitution ◽  
Oxoglutarate Dehydrogenase

Characterization of lysine acetylation in the peripheral subunit-binding domain of the E2 subunit of the pyruvate dehydrogenase-2-oxoglutarate dehydrogenase hybrid complex from Corynebacterium glutamicum

2020 ◽  
Vol 85 (4) ◽  
pp. 874-881
Author(s):  
Ayano Komine-Abe ◽  
Naoko Kondo ◽  
Shosei Kubo ◽  
Hisashi Kawasaki ◽  
Makoto Nishiyama ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Critical Role ◽  
Binding Domain ◽  
Lysine Acetylation ◽  
Hybrid Complex ◽  
Glutamate Production ◽  
Accumulation Kinetic ◽  
Oxoglutarate Dehydrogenase

ABSTRACT In Corynebacterium glutamicum, pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (ODH) form a unique hybrid complex in which CgE1p and CgE1o are associated with the CgE2–CgE3 subcomplex. We analyzed the role of a lysine acetylation site in the peripheral subunit-binding domain of CgE2 in PDH and ODH functions. Acetylation-mimic substitution at Lys391 of CgE2 severely reduced the interaction of CgE2 with CgE1p and CgE3, but not with CgE1o, indicating the critical role of this residue in the assembly of CgE1p and CgE3 into the complex. It also suggested that Lys391 acetylation inhibited the binding of CgE1p and CgE3 to CgE2, thereby affecting PDH and ODH activities. Interestingly, the CgE2-K391R variant strain showed increased l-glutamate production and reduced pyruvate accumulation. Kinetic analysis suggested that the increased affinity of the K391R variant toward pyruvate might be advantageous for l-glutamate production.

scholarly journals The E2 Domain of OdhA of Corynebacterium glutamicum Has Succinyltransferase Activity Dependent on Lipoyl Residues of the Acetyltransferase AceF

2010 ◽  
Vol 192 (19) ◽  
pp. 5203-5211 ◽  
Author(s):  
Melanie Hoffelder ◽  
Katharina Raasch ◽  
Jan van Ooyen ◽  
Lothar Eggeling
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
The Other ◽  
Central Metabolism ◽  
Mutant Proteins ◽  
Oxoglutarate Dehydrogenase ◽  
The Individual ◽  
Activity Dependent ◽  
Dihydrolipoyl Dehydrogenase ◽  
Mutant Cells

ABSTRACT Oxoglutarate dehydrogenase (ODH) and pyruvate dehydrogenase (PDH) complexes catalyze key reactions in central metabolism, and in Corynebacterium glutamicum there is indication of an unusual supercomplex consisting of AceE (E1), AceF (E2), and Lpd (E3) together with OdhA. OdhA is a fusion protein of additional E1 and E2 domains, and odhA orthologs are present in all Corynebacterineae, including, for instance, Mycobacterium tuberculosis. Here we show that deletion of any of the individual domains of OdhA in C. glutamicum resulted in loss of ODH activity, whereas PDH was still functional. On the other hand, deletion of AceF disabled both PDH activity and ODH activity as well, although isolated AceF protein had solely transacetylase activity and no transsuccinylase activity. Surprisingly, the isolated OdhA protein was inactive with 2-oxoglutarate as the substrate, but it gained transsuccinylase activity upon addition of dihydrolipoamide. Further enzymatic analysis of mutant proteins and mutant cells revealed that OdhA specifically catalyzes the E1 and E2 reaction to convert 2-oxoglutarate to succinyl-coenzyme A (CoA) but fully relies on the lipoyl residues provided by AceF involved in the reactions to convert pyruvate to acetyl-CoA. It therefore appears that in the putative supercomplex in C. glutamicum, in addition to dihydrolipoyl dehydrogenase E3, lipoyl domains are also shared, thus confirming the unique evolutionary position of bacteria such as C. glutamicum and M. tuberculosis.

In Vitro Reconstitution and Biophysical Characterization of OEP16, an Outer Envelope Pore Protein of Pea Chloroplasts†

Biochemistry ◽  
2000 ◽  
Vol 39 (36) ◽  
pp. 11050-11056 ◽  
Author(s):  
Dirk Linke ◽  
Joachim Frank ◽  
Josef F. Holzwarth ◽  
Jürgen Soll ◽  
Christoph Boettcher ◽  
...  
Keyword(s):  
Outer Envelope ◽  
Biophysical Characterization ◽  
In Vitro Reconstitution ◽  
Pore Protein

In vitro Reconstitution and Characterization of the Yeast Mitochondrial Degradosome Complex Unravels Tight Functional Interdependence

2007 ◽  
Vol 372 (1) ◽  
pp. 23-36 ◽  
Author(s):  
Michal Malecki ◽  
Robert Jedrzejczak ◽  
Piotr P. Stepien ◽  
Pawel Golik
Keyword(s):  
In Vitro Reconstitution

scholarly journals Functional characterization of human bitter taste receptors

2007 ◽  
Vol 403 (3) ◽  
pp. 537-543 ◽  
Author(s):  
Eduardo Sainz ◽  
Margaret M. Cavenagh ◽  
Joanne Gutierrez ◽  
James F. Battey ◽  
John K. Northup ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Bitter Taste ◽  
Aristolochic Acid ◽  
Functional Characterization ◽  
Gene Families ◽  
In Vitro Reconstitution ◽  
Human And Mouse

The T2Rs belong to a multi-gene family of G-protein-coupled receptors responsible for the detection of ingested bitter-tasting compounds. The T2Rs are conserved among mammals with the human and mouse gene families consisting of about 25 members. In the present study we address the signalling properties of human and mouse T2Rs using an in vitro reconstitution system in which both the ligands and G-proteins being assayed can be manipulated independently and quantitatively assessed. We confirm that the mT2R5, hT2R43 and hT2R47 receptors respond selectively to micromolar concentrations of cycloheximide, aristolochic acid and denatonium respectively. We also demonstrate that hT2R14 is a receptor for aristolochic acid and report the first characterization of the ligand specificities of hT2R7, which is a broadly tuned receptor responding to strychnine, quinacrine, chloroquine and papaverine. Using these defined ligand–receptor interactions, we assayed the ability of the ligand-activated T2Rs to catalyse GTP binding on divergent members of the Gα family including three members of the Gαi subfamily (transducin, Gαi1 and Gαo) as well as Gαs and Gαq. The T2Rs coupled with each of the three Gαi members tested. However, none of the T2Rs coupled to either Gαs or Gαq, suggesting the T2Rs signal primarily through Gαi-mediated signal transduction pathways. Furthermore, we observed different G-protein selectivities among the T2Rs with respect to both Gαi subunits and Gβγ dimers, suggesting that bitter taste is transduced by multiple G-proteins that may differ among the T2Rs.

scholarly journals Subcellular Localization and Characterization of the ParAB System from Corynebacterium glutamicum

2010 ◽  
Vol 192 (13) ◽  
pp. 3441-3451 ◽  
Author(s):  
Catriona Donovan ◽  
Astrid Schwaiger ◽  
Reinhard Krämer ◽  
Marc Bramkamp
Keyword(s):  
Subcellular Localization ◽  
Corynebacterium Glutamicum ◽  
Consensus Sequence ◽  
Polar Localization ◽  
Daughter Cells ◽  
Division Site ◽  
Opposite Pole ◽  
Segregation Of Chromosomes

ABSTRACT Faithful segregation of chromosomes and plasmids is a vital prerequisite to produce viable and genetically identical progeny. Bacteria use a specialized segregation system composed of the partitioning proteins ParA and ParB to segregate certain plasmids. Strikingly, homologues of ParA and ParB are found to be encoded in many chromosomes. Although mutations in the chromosomal Par system have effects on segregation efficiency, the exact mechanism by which the chromosomes are segregated into the daughter cells is not fully understood. We describe the polar localization of the ParB origin nucleoprotein complex in the actinomycete Corynebacterium glutamicum. ParB and the origin of replication were found to be stably localized to the cell poles. After replication, the origins move toward the opposite pole. Purified ParB was able to bind to the parS consensus sequence in vitro. C. glutamicum possesses two ParA-like partitioning ATPase proteins. Both proteins interact with ParB but show a slightly different subcellular localization and phenotype. While ParA might be part of a conventional partitioning system, PldP seems to play a role in division site selection.

Sign in / Sign up

Export Citation Format

Share Document