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  • Shah, R.; Large, A.T.; Ursinus, A.; Lin, B.; Gowrinathan, P.; Martin, J.; Lund, P.A.
    Replacement of GroEL in Escherichia coli by the group II chaperonin from the archaeon Methanococcus maripaludis (2016), J. Bacteriol., 198, 2692-2700 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.6.4.B10 recombinant expression of archaeal group II chaperonin Mm-cpn in Escherichia coli strain TAB21 lacking its group I chaperonin GroEL, and wild-type and mutant enzymes overexpression in Escherichia coli strain BL21(DE3) Methanococcus maripaludis

Protein Variants

EC Number Protein Variants Comment Organism
3.6.4.B10 D386A site-directed mutagenesis, introduction of D386A into Mm-cpn significantly reduces its ability to complement for loss of GroES and GroEL, loss of ATPase activity severely affects the complementing ability of the wild-type and mutant Mm-cpn proteins Methanococcus maripaludis
3.6.4.B10 K216A site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216C site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216D site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216E random mutagenesis, growth for the mutants is clearly faster than for wild-type Mm-cpn organisms under GroES/GroEL-limiting conditions, improved phenotype in Escherichia coli under GroEL- and GroES-depleting conditions. The mutant can effectively hydrolyze ATP Methanococcus maripaludis
3.6.4.B10 K216E/D386A site-directed mutagenesis, introduction of D386A into Mm-cpn significantly reduces its ability to complement for loss of GroES and GroEL, loss of ATPase activity severely affects the complementing ability of the wild-type and mutant Mm-cpn proteins Methanococcus maripaludis
3.6.4.B10 K216F site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216G site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216L site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216P site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216Q site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216R site-directed mutagenesis, the mutant enzyme slightly complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216S site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216T site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 K216V site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 K216Y site-directed mutagenesis, the mutant enzyme moderately complements the GroEL-deletion mutant Escherichia coli strain TAB21 Methanococcus maripaludis
3.6.4.B10 M223E site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223F site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223G site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223I random mutagenesis, growth for the mutants is clearly faster than for wild-type Mm-cpn organisms under GroES/GroEL-limiting conditions, improved phenotype in Escherichia coli under GroEL- and GroES-depleting conditions. The mutant can effectively hydrolyze ATP Methanococcus maripaludis
3.6.4.B10 M223I site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223I/D386A site-directed mutagenesis, introduction of D386A into Mm-cpn significantly reduces its ability to complement for loss of GroES and GroEL, loss of ATPase activity severely affects the complementing ability of the wild-type and mutant Mm-cpn proteins Methanococcus maripaludis
3.6.4.B10 M223L site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223R site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223S site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223V site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223W site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 M223Y site-directed mutagenesis, the mutant enzyme complements the GroEL-deletion mutant Escherichia coli strain TAB21 well Methanococcus maripaludis
3.6.4.B10 additional information summary of the functional growth analysis of Escherichia coli TAB21 cells expressing diverse Mm-cpn-M223 and Mm-cpn-K216 mutants at 30°C, overview. The Mm-cpn-K216E and Mm-cpn-M223I mutants act as genuine chaperonins and must complete an ATP-dependent chaperonin cycle to function in Escherichia coli Methanococcus maripaludis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.6.4.B10 Mg2+ required Methanococcus maripaludis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.6.4.B10 900000
-
 about, Mm-cpn protein complex, gel filtration and native PAGE Methanococcus maripaludis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.6.4.B10 ATP + H2O Methanococcus maripaludis
-
 ADP + phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.6.4.B10 Methanococcus maripaludis Q877G8 i.e. Methanococcus deltae
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.6.4.B10 recombinant chaperonin Mm-cpn from Escherichia coli strain BL21(DE3) by anion exchange chromatography, ammonium sulfate fractionation, and gel filtation, followed by ultrafiltration Methanococcus maripaludis

Source Tissue

EC Number Source Tissue Comment Organism Textmining
3.6.4.B10 additional information the optimum growth temperature of the parent organism is 35-40°C Methanococcus maripaludis
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.6.4.B10 ATP + H2O
-
 Methanococcus maripaludis ADP + phosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.6.4.B10 More Mm-cpn is homooligomeric Methanococcus maripaludis

Synonyms

EC Number Synonyms Comment Organism
3.6.4.B10 archaeal chaperonin
-
 Methanococcus maripaludis
3.6.4.B10 group II chaperonin
-
 Methanococcus maripaludis
3.6.4.B10 Mm-cpn
-
 Methanococcus maripaludis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.6.4.B10 37
-
 ATPase assay at Methanococcus maripaludis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.6.4.B10 7.5
-
 ATPase assay at Methanococcus maripaludis

General Information

EC Number General Information Comment Organism
3.6.4.B10 malfunction the wild-type organism shows reduced growth under GroES/GroEL-limiting conditions. Cells are transformed with plasmids expressing Mm-cpn, Mm-cpn-M223I, and Mm-cpn-K216E and grown at 26°C, 30°C, and 37°C in the presence of sucrose. Apart from the positive control, growth is observed only for cells expressing the mutant Mm-cpn-K216E. These cells show distinct visible colonies after 3 days at 26°C and 30°C in numbers comparable to those observed for cells expressing GroEL. Cells expressing Mm-cpn-K216E also form visible colonies at 37°C after 5 days. Loss of ATPase activity by mutation D386A severely affects the complementing ability of the wild-type and mutant Mm-cpn proteins Methanococcus maripaludis
3.6.4.B10 additional information ATP hydrolysis is important to attain the closed conformation of the enzyme Methanococcus maripaludis