Sterol C -methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition

Mangla, Anil T.; Nes, W. David

doi:10.1016/s0968-0896(00)00040-7

Cited by 39 publications

(47 citation statements)

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“…In work with the yeast SMT, 25-azalanosterol was discovered to bind to the active center with a K d value of 4 µM similar to either the K d values of zymosterol or AdoMet at 4 µM [20]. The expectation for tight binding was borne out by the results of the 24-and 25-heteroatom substituted sterols assayed with SMT1 and SMT2, the carbocation mimic was bound to the Michaelis complex many orders more tightly than the sterol (or AdoMet) substrate generating a K m /K i ratio of approximately 1000 [16,23,38,[40][41][42][43][44][45][46]. The efficacy of 25-azacycloartenol, a substrate mimic, and solasodine have been compared in vitro and in vivo with the pathogenic yeastlike microbe P. wickerhamii which synthesizes ergosterol by the cycloartenol-cyclolaudenol route shown in Scheme 1.…”

Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 96%

“…2.1.1.142) and 24(28)-methylenelophenol is the preferred substrate of the plant SMT2 (E.C. 2.1.1.143) [6,14,18,[21][22][23].…”

Section: Smt Propertiesmentioning

confidence: 99%

“…Alternatively, the SMTs are appealing targets for the design of enzyme inhibitors, since they lie on a pathway that is key for the synthesis of membrane inserts in microbes and plants and the unique aspects of SMT structure and function make for the design of species-specific inhibitors that will not be harmful to mammalian physiology [6]. Evidence that SMT action can be impaired by sterol analogs with an ammonium function in the side chain prepared synthetically or from nature (Scheme 4) [22,23,28,39] prompted us to take a detailed analysis of SMT inhibitors and to prepare and test a range of diverse structures. It is assumed that the inhibitory power of the compounds in Scheme 4 is related to the tertiary amine function of these molecules which will be protonated at physiological pH and that therefore can mimic the high energy intermediate in the C-methylation reaction progress [22].…”

Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 99%

“…Reversible inhibitors can be classified based on whether they are (i) high energy intermediate analogs or (ii) dead-end analogs, and the irreversible inhibitors can be classified as (iii) mechanismbased inactivators. The compounds shown in Scheme 4 have been tested and found to be noncompetitive inhibitors against the native substrate of the SMT [22,23,28,39]. In spite of numerous side chain modifications [38,[40][41][42][43][44][45][46][47], only a limited number of structural variants exhibit binding affinities to the enzyme active site that are significantly greater than those of the native substrate.…”

Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 99%

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Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

et al. 2004

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Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.

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Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 96%

“…2.1.1.142) and 24(28)-methylenelophenol is the preferred substrate of the plant SMT2 (E.C. 2.1.1.143) [6,14,18,[21][22][23].…”

Section: Smt Propertiesmentioning

confidence: 99%

Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 99%

Section: Rational Design Of Potent Inhibitors Of Smtmentioning

confidence: 99%

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Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

et al. 2004

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“…DISCUSSION Generally, the kinetic constants of SMT have not been well characterized. The V max values reported seem to refer to enzyme activities and therefore to the corresponding variations in the amounts of the enzyme in different plants or fungi rather than to fundamental kinetic parameters of the enzyme reactions (10,14,19,26,33,34,36,37,39). The inability to properly characterize SMTs in these organisms also stems from not assaying the optimal substrate for a particular isoform, thereby underestimating the number of SMT isoforms present in the plant and complicating interpretation of the activity assays.…”

Section: Conversion Of 28-2 H Substrates To 24-methylatedmentioning

confidence: 99%

Biosynthesis of Phytosterols

Nes

Song

Dennis

et al. 2003

Journal of Biological Chemistry

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Cloned soybean sterol methyltransferase was purified from Escherichia coli to gel electrophoretic homogeneity. From initial velocity experiments, catalytic constants for substrates best suited for the first and second C 1 transfer activities, cycloartenol and 24(28)-methylenelophenol, were 0.01 and 0.001 s ؊1 , respectively. Two-substrate kinetic analysis using cycloartenol and S-adenosyl-L-methionine (AdoMet) generated an intersecting line pattern characteristic of a ternary complex kinetic mechanism. H 3 ]AdoMet to examine the kinetic isotope effects attending the C-28 deprotonation in the enzymatic synthesis of 24-ethyl(idene) sterols. The stereochemical features as well as the observation of isotopically sensitive branching during the second C-methylation suggests that the two methylation steps can proceed by a change in chemical mechanism resulting from differences in sterol structure, concerted versus carbocation; the kinetic mechanism remains the same during the consecutive methylation of the ⌬ 24 bond.Sterol methyltransferases (SMTs) 1 are ubiquitously represented in plants and fungi (1). Together, these enzymes generate 24-alkyl sterol diversity, which includes formation of singly and doubly C-24-alkylated sterol side chains and olefin variants possessing ⌬ 24(28) , ⌬ 23 (24) , and ⌬ 25(27) side chains (2). In most organisms, SMTs catalyze the first committed step in the biosynthesis of phytosterols (3, 4) (Fig. 1). The crucial role of these enzymes to generate an essential physiological group in sterol structure has stimulated considerable interest in the stereochemistry and mechanism of the C-methylation reaction (5, 6). Several SMTs have been characterized at the molecular level, and their amino acid compositions reveal a highly conserved signature motif that represents the sterol-binding site (7,8). A number of these enzymes have been characterized, and they share similar native molecular masses in the range of 160 -175 kDa and similar properties (1). The methyl transfer reaction catalyzed by SMT is proposed to proceed through a nucleophilic attack by the electrons of the ⌬ 24 double bond on the S-methyl group of AdoMet (9 -11). The reaction can lead to the formation of a high energy intermediate (HEI) possessing a methyl at C-24 and a carbonium ion at C-25. After a hydride transfer from C-24 to C-25, an elimination of a proton at C-28 occurs, giving a 24(28)-methylene sterol. The steric course of the reaction has been hypothesized to proceed by an "X-group" (covalent), carbocation, or concerted mechanism (Scheme 1) (8).As recognized in the steric-electric plug model and X-group mechanism, the conformation of the bound sterol side chain can influence the configuration of the enzyme-generated product at C-24 and C-25 (Scheme 1A) (8).Recent work on the stereochemistry of phytosterol (24-alkyl sterols) side chain carbon atoms harboring chemically or biosynthetically introduced 13 C label showed that the natural configuration for C-26 and C-27 of ergosterol and sitosterol (C-25 S; 2) is opposite to that...

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Protothecosis

Pore

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Sterol C -methyl transferase from Prototheca wickerhamii mechanism, sterol specificity and inhibition

Cited by 39 publications

References 54 publications

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Biosynthesis of Phytosterols

Protothecosis

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