W. David Nes

STEROL METHYLTRANSFERASE 1 Controls the Level of Cholesterol in Plants

The side chain in plant sterols can have either a methyl or ethyl addition at carbon 24 that is absent in cholesterol. The ethyl addition is the product of two sequential methyl additions. Arabidopsis contains three genes—sterol methyltransferase 1 (SMT1), SMT2, and SMT3—homologous to yeast ERG6, which is known to encode an S-adenosylmethionine–dependent C-24 SMT that catalyzes a single methyl addition. The SMT1 polypeptide is the most similar of these Arabidopsis homologs to yeast Erg6p. Moreover, expression of Arabidopsis SMT1 in erg6 restores SMT activity to the yeast mutant. The smt1 plants have pleiotropic defects: poor growth and fertility, sensitivity of the root to calcium, and a loss of proper embryo morphogenesis. smt1 has an altered sterol content: it accumulates cholesterol and has less C-24 alkylated sterols content. Escherichia coli extracts, obtained from a strain expressing the Arabidopsis SMT1 protein, can perform both the methyl and ethyl additions to appropriate sterol substrates, although with different kinetics. The fact that smt1 null mutants still produce alkylated sterols and that SMT1 can catalyze both alkylation steps shows that there is considerable overlap in the substrate specificity of enzymes in sterol biosynthesis. The availability of the SMT1 gene and mutant should permit the manipulation of phytosterol composition, which will help elucidate the role of sterols in animal nutrition.

The Metabolism, Structure, and Function of Plant Lipids

The three types of plant prenyllipids, the chlorophylls, carotenoids and prenylquinones, are integral components of a functional photosynthetic apparatus. The functional organization of these prenyllipids within the photosynthetic membrane is far from being well understood. Our present knowledge of the functional association and partition of individual carotenoids and prenylquinones with pigment proteins is reviewed. The importance of gal actoand phospholipids for a functional integration of pigments and prenylquinones in the photosynthetic membrane is underlined by results with the new grass-herbicide sethoxydim, which inhibits glycerolipid biosynthesis and blocks the accumulation but not the biosynthesis of pigments and prenylquinones. INTRODUCTION Chlorophylls and carotenoids, the photosynthetic prenylpigments and the prenylquinone derivatives plastoquinone-g, phylloquin~ne K1, ~-tocoquinone and o(-tocopherol form the group of functional chloroplast prenyllipids which are located in the thylakoids and are needed to perform the light reactions of photosynthesis (1,2). Many aspects of biosynthesis and functions of these plastidic prenyllipids had been reviewed at preceding plant lipid symposia. This report will therefore concentrate on some recent developments which might give new insights into function or metabolism of these thylakoid components. The three chapters deal with A) chlorophylls and carotenoids, B) prenylquinones, and C) the inhibition of prenyllipid accumulation and acyllipid biosynthesis in chloroplasts by the new grass herbicide sethoxydim. A. CHLOROPHYLLS AND CAROTENOIDS In algae and higher plants it is now well documented that chlorophylls and carotenoids are quantitatively bound within the thylakoids in various ways to different chlorophyll-carotenoid pigment-proteins. By polyacrylamide gel electrophoresis (PAGE) of digitonin-digested or sodium dodecylsulfate-treated thylakoid preparations one obtains 3 types of pigment proteins (3-7):

Insect Sterol Nutrition and Physiology: A Global Overview

Abstract Unlike most animals, insects lack the capacity to synthesize sterols that are required in lipid biostructures, as precursors to important steroid hormones and as regulators of developmental processes. Therefore insects must acquire sterols from their diet. Hundreds of different sterols have been identified and the review starts by documenting the occurrence of sterols in different insect foods. Next we look at the various nutritional and biochemical studies that have been conducted, and organize them according to insect relatedness, which allows insect sterol use and metabolic capabilities to be viewed from an evolutionary perspective. How sterol structure influences insect feeding behavior is examined, and the fate of sterols once they have been ingested, including the processes of absorption and transport, their distribution in different tissues, and their role in reproduction, is detailed. The extent to which sterols may influence ecological outcomes is also considered, especially in phytophagous insects with known sterol metabolic constraints. Finally, mention is made of the potential use of exploiting insect sterol requirements and constraints for pest control, as well as the ability of insects to adapt to the presence of novel sterols in their diet.

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.

CYP51 from Trypanosoma cruzi A PHYLA-SPECIFIC RESIDUE IN THE B′ HELIX DEFINES SUBSTRATE PREFERENCES OF STEROL 14α-DEMETHYLASE

A potential drug target for treatment of Chagas disease, sterol 14α-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from ∼25% between phyla to ∼80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B′ helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14α-demethylation of obtusifoliol and its 24-demethyl analog 4α-,4α-dimethylcholesta-8,24-dien-3β-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced ∼3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites.

Crystal Structures of Trypanosoma brucei Sterol 14α-Demethylase and Implications for Selective Treatment of Human Infections

Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

Comparison of the chromatographic properties of sterols, select additional steroids and triterpenoids: gravity-flow column liquid chromatography, thin-layer chromatography, gas—liquid chromatography and high-performance liquid chromatography

The chromatographic properties of approximately 100 sterols, select steroids of plant origin (sapogenins and steroidal alkaloids) and triterpenoids has been evaluated in this laboratory by monitoring their elution characteristics in adsorption (gravity column and thin-layer methods with and without the addition of silver nitrates), gas and reversed-phase high-performance liquid chromatography. The utility of each methodology to act in one or another chromatographic mode-separation, radio-chemical purification, quantitation and structural elucidation, is discussed. The importance of the tilt of the -OH group at C-3 as well as the polarity, size, an shape of the rest of the molecule as it effects the hydrogen-bonding ability of the -OH group is demonstrated through changes in chromatographic behavior that result from the step-wise introduction of double bonds, methyl, bromo, oxygen, nitrogen and cyclopropyl groups into 5 alpha-cholestanol. An independent aid in the structure identification and quantitation of the compounds was use of a multiple-wavelength diode array detector in which different wavelengths of the UV spectrum (200-400 nm) were simultaneously monitored following passage of the sample through a reversed-phase C18 column.

Carbon-13 nmr studies on sitosterol biosynthesized from [13C]mevalonates

[2-13C]Mevalonic acid and [5-13C]mevalonic acid were fed to cultured cells of sunflower and the resulting biosynthesized [13C]sitosterols were examined by 13C NMR spectroscopy. The observations confirmed the biochemically predictable positions of 13C labels in sitosterol. Based on the new information a mechanism for the biogenesis of the C-24 alkyl group in phytosterols is given.

Identification of the Lipophilic Factor Produced by Macrophages That Stimulates Steroidogenesis1

Macrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC column...

Identification of Natural RORγ Ligands that Regulate the Development of Lymphoid Cells

Mice deficient in the nuclear hormone receptor RORγt have defective development of thymocytes, lymphoid organs, Th17 cells, and type 3 innate lymphoid cells. RORγt binds to oxysterols derived from cholesterol catabolism, but it is not clear whether these are its natural ligands. Here, we show that sterol lipids are necessary and sufficient to drive RORγt-dependent transcription. We combined overexpression, RNAi, and genetic deletion of metabolic enzymes to study RORγ-dependent transcription. Our results are consistent with the RORγt ligand(s) being a cholesterol biosynthetic intermediate (CBI) downstream of lanosterol and upstream of zymosterol. Analysis of lipids bound to RORγ identified molecules with molecular weights consistent with CBIs. Furthermore, CBIs stabilized the RORγ ligand-binding domain and induced coactivator recruitment. Genetic deletion of metabolic enzymes upstream of the RORγt-ligand(s) affected the development of lymph nodes and Th17 cells. Our data suggest that CBIs play a role in lymphocyte development potentially through regulation of RORγt.