Probing the role of the 3-hydroxy group of lanosterol in substrate-enzyme interactions of the Paracoccidioides brasiliensis sterol C24-methyltransferase



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The role of the sterol C3-hydroxyl group of lanosterol in the reaction catalyzed by Paracoccidioides brasiliensis sterol C24-methyltransferase (PbSMT) was studied kinetically and for product differences against a panel of modified lanosterol C3-derivatives in which the natural C3β-OH was chemically replaced with α-OH, OMe, OAc, keto, NH₂, F, H and NH₃⁺. PbSMT catalyzed variably 7 test substrates that retained nucleophilic character whereas neither the 3-desoxy nor the salt analogs were productively bound. GC-MS analysis of the biomethyl products catalyzed by PbSMT showed similar recognition of the sterol side chain; each of them contained the same Δ²⁴⁽²⁸⁾- construction. Alternatively, significant differences in the catalytic competence (Vmax/ Km) of the analogs tested revealed sterol-specific Michaelis complexes resulting from the differences associated with the size, electronics and configuration of the substrate C3-group. These results revealed that polarity and stereochemistry of the C3-group is essential in the enzyme-substrate interactions of PbSMT and that the 3β-OH group is the best structural feature tested for optimal activity.



C24 sterol methyltransferase, Paracoccidioides brasiliensis, C3-OH, Hydrogen bonding