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Literature summary extracted from

  • Upadhyay, V.; Demmer, U.; Warkentin, E.; Moll, J.; Shima, S.; Ermler, U.
    Structure and catalytic mechanism of N5,N10-methenyl-tetrahydromethanopterin cyclohydrolase (2012), Biochemistry, 51, 8435-8443.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.4.27 overexpression in Escherichia coli Archaeoglobus fulgidus

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.4.27 hanging drop vapor diffusion method in an anaerobic tent with an atmosphere of 95% N2/5% H2 under low-intensity red light. X-ray structures of the substrate-free E186Q mutant enzyme, the enzyme:5,10-methenyl-5,6,7,8-tetrahydromethanopterin complex, and the E186Q mutant enzyme:5-formyl-5,6,7,8-tetrahydromethanopterin complex Archaeoglobus fulgidus

Protein Variants

EC Number Protein Variants Comment Organism
3.5.4.27 E186A Vmax is 0.035% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 2.1fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 E186D Vmax is 0.56% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.1fold higher compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 E186N Vmax is 0.1% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.1fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 E186Q nearly inactive mutant enhzyme Archaeoglobus fulgidus
3.5.4.27 F190V no activity Archaeoglobus fulgidus
3.5.4.27 K94E Vmax is 86% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.4fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 K94V Vmax is 41% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.2fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 R183a Vmax is 0.07% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 R183E Vmax is 0.01% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 2.2fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 R183E/E186Q no activity Archaeoglobus fulgidus
3.5.4.27 R183K Vmax is 15% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value Archaeoglobus fulgidus
3.5.4.27 R183Q Vmax is 0.05% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value Archaeoglobus fulgidus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.4.27 0.025
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme R183E Archaeoglobus fulgidus
3.5.4.27 0.026
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme E186A Archaeoglobus fulgidus
3.5.4.27 0.043
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme R183A Archaeoglobus fulgidus
3.5.4.27 0.043
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme R183K Archaeoglobus fulgidus
3.5.4.27 0.043
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme R183Q Archaeoglobus fulgidus
3.5.4.27 0.047
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme K94V Archaeoglobus fulgidus
3.5.4.27 0.05
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme E186N Archaeoglobus fulgidus
3.5.4.27 0.055
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, wild-type enzyme Archaeoglobus fulgidus
3.5.4.27 0.062
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme E186D Archaeoglobus fulgidus
3.5.4.27 0.077
-
 5,10-methenyl-5,6,7,8-tetrahydromethanopterin pH 7.6, 65°C, mutant enzyme K94E Archaeoglobus fulgidus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.4.27 5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O Archaeoglobus fulgidus
-
 5-formyl-5,6,7,8-tetrahydromethanopterin
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.4.27 Archaeoglobus fulgidus O28344
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.4.27
-
 Archaeoglobus fulgidus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.4.27 5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
-
 Archaeoglobus fulgidus 5-formyl-5,6,7,8-tetrahydromethanopterin
-
 ?
3.5.4.27 5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O the active site is primarily built up by the segment 93:95, Arg183 and Glu186 that either interact with the catalytic water attacking 5,10-methenyl-5,6,7,8-tetrahydromethanopterin or with the formyl oxygen of 5-formyl-5,6,7,8-tetrahydromethanopterin. The catalytic function of the strictly conserved Arg183 and Glu186 is substantiated by the low enzymatic activities of the E186A, E186D, E186N, E186Q, R183A, R183Q, R183E, R183K, and R183E-E186Q variants. Glu186 most likely acts as a general base. Arg183 decisively influences the pKa value of Glu186 and the proposed catalytic water mainly by its positive charge. In addition, Glu186 appears to be also responsible for product specificity by donating a proton to the directly neighbored N10 tertiary amine of H4MPT. Thus, N10 becomes a better leaving group than N5 which implies the generation of 5-formyl-5,6,7,8-tetrahydromethanopterin Archaeoglobus fulgidus 5-formyl-5,6,7,8-tetrahydromethanopterin
-
 ?

Synonyms

EC Number Synonyms Comment Organism
3.5.4.27 Mch
-
 Archaeoglobus fulgidus
3.5.4.27 methenyl-H4MPT+ cyclohydrolase
-
 Archaeoglobus fulgidus
3.5.4.27 methenyltetrahydromethanopterin cyclohydrolase
-
 Archaeoglobus fulgidus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.4.27 65
-
 assay at Archaeoglobus fulgidus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.4.27 7.6
-
 assay at Archaeoglobus fulgidus

General Information

EC Number General Information Comment Organism
3.5.4.27 physiological function the enzyme catalyzes a reaction in the one-carbon energy metabolism of methanogenic, methanotrophic, and sulfate-reducing archaea and of methylotrophic bacteria Archaeoglobus fulgidus