Sterol methyltransferase: Probing its drug-target and allosteric properties
Patkar, Presheet P.
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The methylation of cycloartenol by plant 24-SMT (Glycine max) leads to formation of a single product – 24(28)-methylenecycloartenol. This is in stark contrast to the ones made by green algae that leads to two products of cycloartenol catalysis – cyclolaudenol (∆25(27)-olefin) and 24(28)-methylenecycloartenol. Catalysis of substrate analogues like 26-homocycloartenol or 3B-fluorolanostadiene by GmSMT (SSMT) led to formation of a single 24(28) product as expected. Incubation of a sterol analogue modified at C26 with substituted fluorine led to product conversion in favour of ∆25(27)-olefin product via formation of a bound intermediate (cyclolaudenol cation). A part of the substrate was also covalently bound by the enzyme and upon hydrolysis, was identified as 26-flouro-25-hydroxy-24-methylcycloartenol. These 26-fluoro (26-F) compounds are also potent competitive inhibitors and generated irreversible competitive inhibition with a kinact of 0.12min-1. This capability of GmSMT to catalyze the formation of a 25-hydroxy alkylated sterol and ∆25(27)-olefins which are fluorinated confirms our hypothesis that 1. The formation of a cycloaudenyl cation is an important step for achieving overall reaction rate and 2. The evolution mechanism of sterols has been driven by reaction channels to where in the evolutionary scheme, in algae that produce primarily ∆25(27) products that convert into ergosterol to land plants which lead to formation of sitosterol via generation of 24(28) products. In accordance with rational drug design strategies to target parasite-specific enzymes of ergosterol biosynthesis, responsible for sleeping sickness, we tested sterols that can covalently bind to SMT and prevent the growth of Trypanosoma brucei. In the presence of about 12-20 µM 26,27-dehydrolanosterol, the sterols lead to halving of the T. brucei procyclic or blood stream form growth. This compound is actively converted by the protozoan sterol metabolic machinery to an intermediate that can inactivate the enzyme via catalytic interaction with SMT. Treated cells show a decrease in ergosterol synthesis over time. This is more effective than using conventional therapeutics like azoles that lead to build up of toxic 14-methyl sterols. In T.brucei cells incubated with the substrate analog 26,27-dehydrozymosterol (DHZ), a metabolite of 26,27-dehydrolanosterol (DHL), the sterol methylating enzyme is inactivated as a result of irreversible binding of the intermediate to the active site. This study has potential applications in the field of rational drug design to generate effective strategies to combat tropical diseases that feature causative organisms dependent on ergosterol biosynthesis. Sterol methyltransferases (SMT) is responsible for the C-24 methylation of intermediates in all non-animal sterol biosynthetic pathways contributing largely to the evolution, diversity and ubiquity of sterols. While the catalytic profile and efficiency of SMTs has been extensively studied in-vitro from crude microsomal extracts and cell-based expression systems, enzymatic profiling of purified SMTs has been known to generate data comparable to that of structurally compromised SMT. Previous efforts demonstrated that the co-factor S-Adenosylmethionine modulated the activity of SMT allosterically. Recently, research efforts on partially purified yeast, plant, algal and protozoan SMTs have indicated that the enzyme is responsive to fluctuations in the energy charge (ATP) of the intra cellular environment resulting in regulation of enzyme activity. Proof that the SMTs are functional homo-tetramers suggests that the interaction of Adenosine triphosphate (ATP), S-Adenosylmethionine (SAM) and sterol at the allosteric site(s) may result in the divergent/differential regulation of the methylation of the natural substrates by SMTs. In-vitro kinetic analysis of the partially purified SMT indicated allosteric upregulation of the enzyme by ATP and SAM. Binding studies revealed Kd values of 0.4uM for both ATP and SAM suggesting a competitive allosteric interplay of SAM and ATP. The subsequent modulation results in a 5-10 fold increase in activity (ScSMT in its ATP deficient form and saturated with ATP shows a kcat transition from 0.5 min-1 to 6 min-1, respectively) and a gain-of-function phenomenon triggering the plant and algal SMTs towards an additional methylation step. Here, we investigate the abovementioned allosteric phenomena of this rate limiting enzymatic process in the context of sterol evolution and diversity while highlighting the role of SMTs in sterol biosynthetic pathways.