Cloned (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
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 |
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 |
Y88F | site-directed mutagenesis, the mutation causes a 10% loss in activity compared to the wild-type enzyme | Paracoccidioides brasiliensis |
Y88L | site-directed mutagenesis, the mutation causes a 50% loss in activity compared to the wild-type enzyme | Paracoccidioides brasiliensis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
25-azalanosterol | substrate analogue, competitive-type inhibitor, a potent inhibitor of cell growth promoting lanosterol accumulation and 24-alkyl sterol depletion | Paracoccidioides brasiliensis | |
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] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | steady-state kinetics and thermodynamics of wild-type and mutant enzymes, overview | Paracoccidioides brasiliensis | |
0.038 | - |
lanosterol | pH 7.5, 30°C, recombinant wild-type enzyme | Paracoccidioides brasiliensis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
42502 | - |
x * 42502, sequence calculation | Paracoccidioides brasiliensis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | - |
? | |
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 | ? | - |
? | |
S-adenosyl-L-methionine + cycloartenol | Paracoccidioides brasiliensis | - |
S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol | - |
? | |
S-adenosyl-L-methionine + cycloartenol | Paracoccidioides brasiliensis Pb0, ATCC MYA-826 | - |
S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol | - |
? | |
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 | ? | |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Paracoccidioides brasiliensis | - |
gene PbSMT | - |
Paracoccidioides brasiliensis Pb0, ATCC MYA-826 | - |
gene PbSMT | - |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | ? | - |
? | |
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 | ? | - |
? | |
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 | ? | - |
? | |
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 | ? | - |
? | |
S-adenosyl-L-methionine + 14-methylzymosterol | - |
Paracoccidioides brasiliensis | ? | - |
? | |
S-adenosyl-L-methionine + 26,27-dehydrolanosterol | Formation of a reversibly bound enzymesubstrate 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 | ? | - |
? | |
S-adenosyl-L-methionine + 31-norlanosterol | - |
Paracoccidioides brasiliensis | ? | - |
? | |
S-adenosyl-L-methionine + cycloartenol | - |
Paracoccidioides brasiliensis | S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol | - |
? | |
S-adenosyl-L-methionine + cycloartenol | - |
Paracoccidioides brasiliensis Pb0, ATCC MYA-826 | S-adenosyl-L-homocysteine + (24R)-24-methylcycloart-25-en-3beta-ol | - |
? | |
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 | ? | |
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 | ? | |
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 | ? | |
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 | Comment | Organism |
---|---|---|
? | x * 42502, sequence calculation | Paracoccidioides brasiliensis |
Synonyms | Comment | Organism |
---|---|---|
SMT | - |
Paracoccidioides brasiliensis |
sterol C24-methyltransferase | - |
Paracoccidioides brasiliensis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Paracoccidioides brasiliensis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0023 | - |
lanosterol | pH 7.5, 30°C, recombinant wild-type enzyme | Paracoccidioides brasiliensis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | 8 | - |
Paracoccidioides brasiliensis |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
6.5 | 9 | half-maximal activities at pH 6.5 and pH 9.0 | Paracoccidioides brasiliensis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
S-adenosyl-L-methionine | - |
Paracoccidioides brasiliensis |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | inhibition kinetics, overview | Paracoccidioides brasiliensis | |
0.000014 | - |
25-azalanosterol | pH 7.5, 30°C, recombinant wild-type enzyme | Paracoccidioides brasiliensis | |
0.054 | - |
26,27-dehydrolanosterol | pH 7.5, 30°C, recombinant wild-type enzyme | Paracoccidioides brasiliensis |
IC50 Value | IC50 Value Maximum | Comment | Organism | Inhibitor | Structure |
---|---|---|---|---|---|
0.00003 | - |
pH 7.5, 30°C, recombinant wild-type enzyme | Paracoccidioides brasiliensis | 25-azalanosterol |
General Information | Comment | Organism |
---|---|---|
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 |
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 |
additional information | residue Tyr88 in the native protein can contribute to stabilization of the active-site topography | Paracoccidioides brasiliensis |