De-an Guo

Developmental regulation of sterol biosynthesis inZea mays

Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized by chromatographic and spectral methods fromZea mays (corn). Several plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-14C]mevalonic acid, [2-14C]acetate, and [2-3H]acetate), and stable isotopes (D2O), we discovered that hydroxymethylglutaryl CoA reductase is not “the” rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation of the sterol side chain. Blades synthesized mainly 24α-ethyl-sterols, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an activede novo pathway. Shoots, normally synthesizing (Z)-24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3β-ol. Examination of the mass spectrum and1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originating with acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed.

MECHANISM AND STRUCTURAL REQUIREMENTS FOR TRANSFORMATION OF SUBSTRATES BY THE (S)-ADENOSYL-L-METHIONINE : DELTA 24(25)-STEROL METHYL TRANSFERASE FROM SACCHAROMYCES CEREVISIAE

The mechanism of action and active site of the enzyme (S)-adenosyl-L-methionine:delta 24(25)-sterol methyl transferase (SMT) from Saccharomyces cerevisiae strain GL7 have been probed with AdoMet, (S)-adenosyl-L-homocysteine, a series of 35 sterol substrates as acceptor molecules and a series of 10 substrate and high energy intermediate (HEI) sterol analogues as inhibitors of biomethylation. The SMT was found to be selective for sterol, both regio- and stereochemically. The presence of an unhindered 24,25-bond, an equatorially-oriented polar group at C-3 (which must act as a proton acceptor) attached to a planar nucleus and a freely rotating side chain were obligatory structural features for sterol binding/catalysis; no essential requirement or significant harmful effects could be found for the introduction of an 8(9)-bond, 14 alpha-methyl or 9 beta,19-cyclopropyl group. Alternatively, methyl groups at C-4 prevented productive sterol binding to the SMT. Initial velocity, product inhibition, and dead-end experiments demonstrated a rapid-equilibrium random bi bi mechanism. Deuterium isotope effects developed from SMT assays containing mixtures of [3-3H]zymosterol with AdoMet or [methyl-2H3]AdoMet confirmed the operation of a random mechanism, kappa H/kappa D = 1.3. From this combination of results, the spatial relationship of the sterol substrate to AdoMet could be approximated and the topology of the sterol binding to the SMT thereby formulated.

Sterol utilization and metabolism by Heliothis zea

Heliothis zea (corn earworm), an insect that fails to synthesize sterols de novo, was reared on an artificial diet treated with 18 different sterol supplements. Larvea did not develop on a sterol-less medium. Δ5-Sterols with a hydrogen atom, a methylene group, an E-or Z-ethylidene group, or an α- or β-ethyl group (cholesterol, ostreasterol, isofucosterol, fucosterol, sitosterol, and clionasterol, respectively) at position C-24, and Δ5-sterols doubly substituted in the side chain at C-24 with an α-ethyl group and at C-22 with a double bond (stigmasterol) supported normal larval growth to late-sixth instar (prepupal: maturity). The major sterol isolated from each of these sterol treatments was cholesterol, suggesting that H. zea operates a typical 24-dealkylation pathway. The sterol requirement of H. zea could not be met satisfactorily by derivatives of 3β-cholestanol with a 9β, 19-cyclopropyl group, gem dimethyl group at C-4, a Δ5,7-bond or Δ8-bond, or by side-chain modified sterols that possessed a Δ25(27)-24β-ethyl group, Δ23(24)-24-methyl group, or 24-ethyl group, or Δ24(25)-24-methyl or 24-ethyl group. The major sterol recovered from the larvae (albeit developmentally arrested larvae) treated with a nonutilizable sterol was the test compound. Sterol absorption was related to the degree of sterol utilization. The most effective sterols absorbed by the insect ranged from 27 to 66 μg per insect, whereas the least effective sterols absorbed by the insect ranged from 0.6 to 6 μg per insect. Competition experiments using different proportions of cholesterol and 24-dihydrolanosterol (from 9:1 to 1:9 mixtures) indicated that abnormal development of H. zea may be induced on less than a 1 to 1 mixture of utilizable (cholesterol) to nonutilizable (24-dihydrolanosterol) sterols. The results demonstrate new structural requirements for sterol utilization and metabolism by insects, particularly with respect to the position of double bonds in the side chain and functionalization in the nucleus. The novel sterol specificities observed in this study appear to be associated with the dual role of sterols as membrane inserts (nonmetabolic) and as precursors to the ecdysteroids (metabolic).

Sterol specificity of theSaccharomyces cerevisiae ERG6 gene product expressed inEscherichia coli

TheERG6 gene fromSaccharomyces cerevisiae has been functionally expressed inEscherichia coli, for the first time, yielding a protein that catalyzes the bisubstrate transfer reaction whereby the reactive methyl group from (S)-adenosyl-l-methionine is transferred stereoselectively to C-24 of the sterol side chain. The structural requirements of sterol in binding and catalysis were similar to the native protein fromS. cerevisiae. Inhibition of biomethylation was observed with fecosterol and ergosterol which suggests that ergosterol may function in wild-type yeast as a feedback regulator of sterol biosynthesis.

Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis

[methyl-2H3]Methionine and zymosterol, [27-13C]lanosterol, [24-2H]lanosterol and lanosterol were separately incubated with the sterol auxotroph Saccharomyces cerevisiae strain GL7. Spectral evidence (1H and 13C-NMR) obtained on three different isotopically labeled ergosterol samples indicated that C-28 was derived from AdoMet, H-24 migrated to C-25 and the C-25 hydrogen on (27-)-methyl 13C-labeled ergosterol was introduced from the Re-face to produce the 25R-stereochemistry.

Stereochemical Features of C-methylations on the Path to Δ24(28)-Methylene and Δ24(28)-Ethylidene Sterols: Studies on the Recombinant Phytosterol Methyl Transferase from Arabidopsis thaliana

Using a homogenate prepared from Escherichia coli cells that express the sterol methyl transferase (SMT) gene of Arabidopsis thaliana, migration of the hydrogen atom at C-24 to C-25 from the Re-face of the double bond was demonstrated in the biosynthesis of [27-13C] 24(28)-methylenezymosterol (fecosterol) from [27-13C]zymosterol and the chirality of the C-25 stereocenter (25R) was found to be retained after the stereospecific conversion of [27-13C]24(28)-methylenezymosterol to [27-13C](24(28)Z) -ethylidenecholest-8-en-3β-ol.

Phytosterol biosynthesis: Isotope effects associated with biomethylation formation to 24-alkene sterol isomers

Abstract Changes in 24-alkene sterol product distribution resulting from deuterium substitution on the coenzyme methyl group of AdoMet and on the sterol acceptor molecule at C-23 and C-24 were used to determine kinetic isotope effects for the terminating deprotonations involved in sterol biomethylation catalyzed by (S)-adenosyl-L-methionine-Δ 24 -sterol methyl transferase (SMT) enzyme. By this method 24(28)-methylene cycloartanol and cyclosadol were shown to be synthesized by two different SMT enzymes.

Antifungal Sterol Biosynthesis Inhibitors

During the course of the last decade, the development of SBIs, and particularly sterol biomethylation inhibitors, has been based on the rational design approach. Successful though this approach has been in elucidating sterol biomethylation enzymology, its limitations are becoming apparent from the findings that: (i) 24,25-double bond metabolism gives rise to cholesterol and ergosterol in a mechanistically similar manner, (ii) 25-azasterols are harmful to human physiology, and (iii) side-chain modified sterols designed to inhibit the SMT enzyme in S. cerevisiae may be ineffective or operate by another kinetic mechanism in a related organism, rendering it therapeutically nonuseful. Nevertheless, it may be possible to ultimately capitalize on the unique aspects of sterol biomethylation chemistry and enzymology to design taxa-specific inhibitors. With increased understanding of the structure and function of SMT enzymes in different fungi, it should be possible to prepare novel mechanism-based inactivators to control SMT activity uniquely and with high specific activity.

Synthesis of Rationally Designed Mechanism-Based Inactivators of the (S)-Adenosyl-L-methionine: Δ24(25)− Sterol Methyl Transferase

Abstract A series of 26,27-cyclopropylidine side chain modified sterols were prepared for the first time from the known aldehydes by Wittig olefination with the ylide from cyclopropyltriphenylphosphonium bromide in butyllithium. Two novel by-products were detected; sterols with nine carbon side chains which lack the terminal isopropyl group and with double bonds in positions 23,24 or 24,25. The structures of the compounds were confirmed by a combination of chromatographic (GLC and HPLC) and spectral (IR, 1H, 13C-NMR) methods. #Visiting Professor from the Department of Pharmacognosy, Beijing Medical University, Beijing 100083, China

Mechanistic and Metabolic Studies of Sterol 24,25-Double Bond Reduction in Manduca sexta

Larvae of Manduca sexta were used to obtain a cell-free sterol 24,25-reductase. From the midgut of fifth instar larvae fed a mixture of sitosterol and campesterol a microsome-bound 24,25-sterol reductase was prepared that transformed desmosterol (K,,,, 3 pM), lanosterol (K,,,, 18 pM), and cycloartenol (K,, 33 pM) to cholesterol, 24,25-dihydrolanosterol, and cycloartanol, respectively. With desmosterol as substrate, the microsome-bound enzyme was found to incorporate tritium into cholesterol from 4s-tritium labelled NADPH. [24-H] lanosterol was transformed by larvae to [24-2H124,25-dihydrolanosterol (structure confirmed by mass spectroscopy (MS) and H-nuclear magnetic resonance spectroscopy. A rationally designed inhibitor of 24,25-reductase activity, 24(R,5Jn,25-epiminolanosterol (IL), was assayed and found to be inhibitory with an 150 of 2 FM. IL was supplemented in the diet of M. sexta with either sitosterol or stigmasterol and found to inhibit development (Iso, 60 pprn). The major sterol which accumulated in the IL-treated larvae was desmosterol, confirming the site of inhibition was reduction of the 24,25-bond. IL was converted to [2-?H]IL when fed to the larvae. [2-3H]lanosterol was recovered from fifth instar larvae and its structure confirmed by MS and radiochemical techniques. Q 1996 WiIey-Liss. Inc.