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Literature summary extracted from

  • Yabuta, Y.; Kamei, Y.; Bito, T.; Arima, J.; Yoneda, K.; Sakuraba, H.; Ohshima, T.; Nakano, Y.; Watanabe, F.
    Functional and structural characteristics of methylmalonyl-CoA mutase from Pyrococcus horikoshii (2015), Biosci. Biotechnol. Biochem., 79, 710-717 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.99.2 genes PH1306 and PH0275 encode the large and small subunits, respectively, the genes encoding the large subunit of MCM are located away from those encoding the small subunit, recombinant expression in Escherichia coli strain 215, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Star (DE3) pLysS Pyrococcus horikoshii

Protein Variants

EC Number Protein Variants Comment Organism
5.4.99.2 additional information generation of N-terminal deletion mutant PH1306DELTAN Pyrococcus horikoshii

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
5.4.99.2 Mg2+ required Pyrococcus horikoshii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.99.2 (R)-methylmalonyl-CoA Pyrococcus horikoshii
-
succinyl-CoA
-
r
5.4.99.2 (R)-methylmalonyl-CoA Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
-
succinyl-CoA
-
r

Organism

EC Number Organism UniProt Comment Textmining
5.4.99.2 Pyrococcus horikoshii O74009 AND O58013 large and small subunits
-
5.4.99.2 Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3 O74009 AND O58013 large and small subunits
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.4.99.2 recombinant His6-tagged wild-type and N-terminal deletion mutant of PH1306 from Escherichia coli strain BL21 Star (DE3) pLysS inclusion bodies by heat treatment at 95°C for 5 min, nickel affinity chromatography, ammonium sulfate fractionation, hydrophobic interaction chromatography, anion exchange chromatography, and gel filtration. rPH0275 is produced in the soluble fraction with high efficiency and purified to homogeneity by nickel affinity chromatography. To obtain substantial quantities of soluble rPH1306, the soluble mutant rPH1306DELTAN is used Pyrococcus horikoshii

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
5.4.99.2 0.0021
-
purified recombinant His-tagged enzyme, in presence of cofactor 5'-deoxyadenosylcobalamin, pH 7.5, 37°C Pyrococcus horikoshii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.99.2 (R)-methylmalonyl-CoA
-
Pyrococcus horikoshii succinyl-CoA
-
r
5.4.99.2 (R)-methylmalonyl-CoA
-
Pyrococcus horikoshii ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3 succinyl-CoA
-
r

Subunits

EC Number Subunits Comment Organism
5.4.99.2 heterotetramer alpha2beta2, the large and small subunits assemble in equimolar ratios and form heterotetrameric complexes in the presence of AdoCbl Pyrococcus horikoshii

Synonyms

EC Number Synonyms Comment Organism
5.4.99.2 MCM
-
Pyrococcus horikoshii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.4.99.2 37
-
assay at, succinyl-CoA is degraded above 50°C Pyrococcus horikoshii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.4.99.2 7.5
-
assay at Pyrococcus horikoshii

Cofactor

EC Number Cofactor Comment Organism Structure
5.4.99.2 5'-deoxyadenosylcobalamin the 5'-deoxyadenosyl vitamin B12 orAdoCbl, methylmalonyl-CoA mutase (MCM) requires 5'-deoxyadenosylcobalamin as a cofactor Pyrococcus horikoshii

General Information

EC Number General Information Comment Organism
5.4.99.2 evolution the two genes PH1306 and PH0275, which encode the subunits of methylmalonyl-CoA mutase in Pyrococcus horikoshii have deduced amino acid sequences that share high homology with the N- and C-terminal regions of human MCM (UniProt ID P22033), respectively. The AdoCbl-binding domain contains a signature DXHXXG motif that is conserved in PH0275. Furthermore, Tyr110, Arg228, and His265 of human MCM are involved in catalysis, and the corresponded amino acid residues are conserved in gene PH1306 Pyrococcus horikoshii
5.4.99.2 physiological function methylmalonyl-CoA mutase catalyzes the reversible interconversion of (2R)-methylmalonyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA), a key intermediate of the tricarboxylic acid cycle. The enzyme requires 5'-deoxyadenosylcobalamin (5'-deoxyadenosyl vitamin B12 or AdoCbl) as a cofactor and is widely distributed in a variety of organisms from bacteria to humans Pyrococcus horikoshii