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Literature summary extracted from

  • Pereira, M.; Song, Z.; Santos-Silva, L.K.; Richards, M.H.; Nguyen, T.T.; Liu, J.; de Almeida Soares, C.M.; da Silva Cruz, A.H.; Ganapathy, K.; Nes, W.D.
    Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis (2010), Biochim. Biophys. Acta, 1801, 1163-1174.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.1.1.41 expressed as a recombinant fusion protein in Escherichia coli BL21 Paracoccidioides brasiliensis
2.1.1.142 gene PbSMT, DNA and amino acid sequence determination and analysis, phylogenetic analysis, overexpression of soluble His-tagged wild-type and mutant enzymes in Escherichia coli strains BL21(DE3) and BL21(C43) Paracoccidioides brasiliensis

Protein Variants

EC Number Protein Variants Comment Organism
2.1.1.142 Y81F site-directed mutagenesis, the mutation causes a preferential change in affinity for DELTA24-sterols that affords alter partitioning in the direction of 24-ethyl(idene) product formation Paracoccidioides brasiliensis
2.1.1.142 Y81L site-directed mutagenesis, the mutation causes a preferential change in affinity for DELTA24-sterols that affords alter partitioning in the direction of 24-ethyl(idene) product formation Paracoccidioides brasiliensis
2.1.1.142 Y88F site-directed mutagenesis, the mutation causes a 10% loss in activity compared to the wild-type enzyme Paracoccidioides brasiliensis
2.1.1.142 Y88L site-directed mutagenesis, the mutation causes a 50% loss in activity compared to the wild-type enzyme Paracoccidioides brasiliensis

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.1.1.41 25-azalanosterol
-
 Paracoccidioides brasiliensis
2.1.1.41 26,27-dehydrolanosterol
-
 Paracoccidioides brasiliensis
2.1.1.142 25-azalanosterol substrate analogue, competitive-type inhibitor, a potent inhibitor of cell growth promoting lanosterol accumulation and 24-alkyl sterol depletion Paracoccidioides brasiliensis
2.1.1.142 26,27-dehydrolanosterol substrate analogue, competitive-type inhibitor, pseudofirst-order, time-dependent inactivation of the PbSMT. Formation of a reversibly bound enzyme-substrate complex, followed by catalysis to an intermediate that can be converted to a methyl product or the intermediate can be intercepted through covalent modification and hence irreversible inhibition, lanosterol or 26,27-dehydrolanosterol can lead to distinct intermediates that convert to methylated sterol products Paracoccidioides brasiliensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.1.1.41 0.019
-
 26,27-dehydrolanosterol pH 7.5, 37°C, Vmax: 6 pmol/min Paracoccidioides brasiliensis
2.1.1.41 0.03
-
 Zymosterol pH 7.5, 37°C, Vmax 2 pmol/min Paracoccidioides brasiliensis
2.1.1.41 0.035
-
 31-norlanosterol pH 7.5, 37°C, Vmax 45 pmol/min Paracoccidioides brasiliensis
2.1.1.41 0.038
-
 lanosterol pH 7.5, 37°C, Vmax: 50 pmol/min Paracoccidioides brasiliensis
2.1.1.142 additional information
-
 additional information steady-state kinetics and thermodynamics of wild-type and mutant enzymes, overview Paracoccidioides brasiliensis
2.1.1.142 0.038
-
 lanosterol pH 7.5, 30°C, recombinant wild-type enzyme Paracoccidioides brasiliensis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.1.1.41 42500
-
 calculated from cDNA Paracoccidioides brasiliensis
2.1.1.142 42502
-
 x * 42502, sequence calculation Paracoccidioides brasiliensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.1.1.142 additional information Paracoccidioides brasiliensis GC-MS analysis shows that the fungus synthesizes 12 compounds of which lanosterol, ergosterol and brassicasterol make up approximately 80% of the sterol mixture ?
-
 ?
2.1.1.142 additional information Paracoccidioides brasiliensis Pb0, ATCC MYA-826 GC-MS analysis shows that the fungus synthesizes 12 compounds of which lanosterol, ergosterol and brassicasterol make up approximately 80% of the sterol mixture ?
-
 ?
2.1.1.142 S-adenosyl-L-methionine + cycloartenol Paracoccidioides brasiliensis
-
 S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol
-
 ?
2.1.1.142 S-adenosyl-L-methionine + cycloartenol Paracoccidioides brasiliensis Pb0, ATCC MYA-826
-
 S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol
-
 ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol Paracoccidioides brasiliensis PbSMT synthesizes a single product, eburicol 24(28)-methylene-24,25-dihydro-lanosterol, from lanosterol S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol Paracoccidioides brasiliensis Pb0, ATCC MYA-826 PbSMT synthesizes a single product, eburicol 24(28)-methylene-24,25-dihydro-lanosterol, from lanosterol S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?

Organism

EC Number Organism UniProt Comment Textmining
2.1.1.41 Paracoccidioides brasiliensis
-
-
-
2.1.1.142 Paracoccidioides brasiliensis
-
 gene PbSMT
-
2.1.1.142 Paracoccidioides brasiliensis Pb0, ATCC MYA-826
-
 gene PbSMT
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.1.1.41 using Ni-NTA chromatography Paracoccidioides brasiliensis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.1.1.41 S-adenosyl-L-methionine + 26,27-dehydrolanosterol
-
 Paracoccidioides brasiliensis ?
-
 ?
2.1.1.41 S-adenosyl-L-methionine + 31-norlanosterol
-
 Paracoccidioides brasiliensis ?
-
 ?
2.1.1.41 S-adenosyl-L-methionine + cycloartenol
-
 Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + 24-methylenecycloartanol
-
 ?
2.1.1.41 S-adenosyl-L-methionine + lanosterol
-
 Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + 24-methylene-24,25-dihydrolanosterol
-
 ?
2.1.1.41 S-adenosyl-L-methionine + zymosterol
-
 Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + fecosterol
-
 ?
2.1.1.142 additional information GC-MS analysis shows that the fungus synthesizes 12 compounds of which lanosterol, ergosterol and brassicasterol make up approximately 80% of the sterol mixture Paracoccidioides brasiliensis ?
-
 ?
2.1.1.142 additional information SMT recognition of lanosterol and cycloartenol versus zymosterol is the C3-OH group, whose orientation in the A-ring and hydrogen bonding ability can affect productive binding of the acceptor molecule. The recombinant enzyme expressed in Escherichia coli possesess a substrate specificity for lanosterol and generates a single exocyclic methylene product. Regiospecific conversion of the pro-Z methyl group of the DELTA24(25)-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation, NMR and mass spectrometric analysis, overview. No activity with 24(28)-methylenelophenol, fecosterol, or eburicol Paracoccidioides brasiliensis ?
-
 ?
2.1.1.142 additional information GC-MS analysis shows that the fungus synthesizes 12 compounds of which lanosterol, ergosterol and brassicasterol make up approximately 80% of the sterol mixture Paracoccidioides brasiliensis Pb0, ATCC MYA-826 ?
-
 ?
2.1.1.142 additional information SMT recognition of lanosterol and cycloartenol versus zymosterol is the C3-OH group, whose orientation in the A-ring and hydrogen bonding ability can affect productive binding of the acceptor molecule. The recombinant enzyme expressed in Escherichia coli possesess a substrate specificity for lanosterol and generates a single exocyclic methylene product. Regiospecific conversion of the pro-Z methyl group of the DELTA24(25)-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation, NMR and mass spectrometric analysis, overview. No activity with 24(28)-methylenelophenol, fecosterol, or eburicol Paracoccidioides brasiliensis Pb0, ATCC MYA-826 ?
-
 ?
2.1.1.142 S-adenosyl-L-methionine + 14-methylzymosterol
-
 Paracoccidioides brasiliensis ?
-
 ?
2.1.1.142 S-adenosyl-L-methionine + 26,27-dehydrolanosterol Formation of a reversibly bound enzyme–substrate complex, followed by catalysis to an intermediate that can be converted to a methyl product or the intermediate can be intercepted through covalent modification and hence irreversible inhibition, lanosterol or 26,27-dehydrolanosterol can lead to distinct intermediates that convert to methylated sterol products Paracoccidioides brasiliensis ?
-
 ?
2.1.1.142 S-adenosyl-L-methionine + 31-norlanosterol
-
 Paracoccidioides brasiliensis ?
-
 ?
2.1.1.142 S-adenosyl-L-methionine + cycloartenol
-
 Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol
-
 ?
2.1.1.142 S-adenosyl-L-methionine + cycloartenol
-
 Paracoccidioides brasiliensis Pb0, ATCC MYA-826 S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol
-
 ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol PbSMT synthesizes a single product, eburicol 24(28)-methylene-24,25-dihydro-lanosterol, from lanosterol Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol PbSMT catalysis of the sterol acceptor lead to a C24-methyl product in which the 1,2-hydride migration of C24 to C25 occurs specifically from the Re-face of the original substrate double bond undergoing transalkylation Paracoccidioides brasiliensis S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol PbSMT synthesizes a single product, eburicol 24(28)-methylene-24,25-dihydro-lanosterol, from lanosterol Paracoccidioides brasiliensis Pb0, ATCC MYA-826 S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?
2.1.1.142 S-adenosyl-L-methionine + lanosterol PbSMT catalysis of the sterol acceptor lead to a C24-methyl product in which the 1,2-hydride migration of C24 to C25 occurs specifically from the Re-face of the original substrate double bond undergoing transalkylation Paracoccidioides brasiliensis Pb0, ATCC MYA-826 S-adenosyl-L-homocysteine + 24(28)-methylene-24,25-dihydro-lanosterol i.e. eburicol ?

Subunits

EC Number Subunits Comment Organism
2.1.1.142 ? x * 42502, sequence calculation Paracoccidioides brasiliensis

Synonyms

EC Number Synonyms Comment Organism
2.1.1.41 24-SMT
-
 Paracoccidioides brasiliensis
2.1.1.41 sterol C24-methyltransferase
-
 Paracoccidioides brasiliensis
2.1.1.142 SMT
-
 Paracoccidioides brasiliensis
2.1.1.142 sterol C24-methyltransferase
-
 Paracoccidioides brasiliensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.1.1.41 30
-
-
 Paracoccidioides brasiliensis
2.1.1.142 30
-
 assay at Paracoccidioides brasiliensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.1.1.41 0.0023
-
 lanosterol pH 7.5, 37°C Paracoccidioides brasiliensis
2.1.1.142 0.0023
-
 lanosterol pH 7.5, 30°C, recombinant wild-type enzyme Paracoccidioides brasiliensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.1.1.41 7.5
-
 assay at Paracoccidioides brasiliensis
2.1.1.142 7.5 8
-
 Paracoccidioides brasiliensis

pH Range

EC Number pH Minimum pH Maximum Comment Organism
2.1.1.41 6.5
-
 half maximum velocities at 6.5 and 9.0 Paracoccidioides brasiliensis
2.1.1.41 7.5 8 optimum pH Paracoccidioides brasiliensis
2.1.1.41 9
-
 half maximum velocities at 6.5 and 9.0 Paracoccidioides brasiliensis
2.1.1.142 6.5 9 half-maximal activities at pH 6.5 and pH 9.0 Paracoccidioides brasiliensis

Cofactor

EC Number Cofactor Comment Organism Structure
2.1.1.142 S-adenosyl-L-methionine
-
 Paracoccidioides brasiliensis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
2.1.1.41 0.000014
-
 25-azalanosterol pH 7.5, 37°C Paracoccidioides brasiliensis
2.1.1.41 0.000054
-
 26,27-dehydrolanosterol pH 7.5, 37°C Paracoccidioides brasiliensis
2.1.1.142 additional information
-
 additional information inhibition kinetics, overview Paracoccidioides brasiliensis
2.1.1.142 0.000014
-
 25-azalanosterol pH 7.5, 30°C, recombinant wild-type enzyme Paracoccidioides brasiliensis
2.1.1.142 0.054
-
 26,27-dehydrolanosterol pH 7.5, 30°C, recombinant wild-type enzyme Paracoccidioides brasiliensis

IC50 Value

EC Number IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
2.1.1.41 0.000014
-
 pH 7.5, 37°C Paracoccidioides brasiliensis 25-azalanosterol
2.1.1.142 0.00003
-
 pH 7.5, 30°C, recombinant wild-type enzyme Paracoccidioides brasiliensis 25-azalanosterol

General Information

EC Number General Information Comment Organism
2.1.1.142 evolution due to differences in its overall amino acid composition and substrate-dependent partitioning pathways, Paracoccidiodes brasiliensis SMT has to be grouped into a fourth and new class of SMT Paracoccidioides brasiliensis
2.1.1.142 metabolism the enzyme is involved in the first metabolic step in the 24-alkyl sterol synthetic pathway of Paracoccidiodes brasiliensis involves the C24-methylation of lanosterol. PbSMT cannot perform the second alkylation step, consistent with the absence of sterols 24(28)-methylenelophenol, fecosterol or eburicol in wild-type cells. Alternate pathways and mechanisms for the conversion of ?24(25)-containing sterol side chains to methylated product, detailed overview Paracoccidioides brasiliensis
2.1.1.142 additional information residue Tyr88 in the native protein can contribute to stabilization of the active-site topography Paracoccidioides brasiliensis