RESUMEN
Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.
Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Calcio , Núcleo Celular , Retículo Endoplásmico , Células Epiteliales , Glándulas Mamarias Animales , Mycoplasma bovis , Animales , Bovinos , Femenino , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Calcio/metabolismo , Monóxido de Carbono/metabolismo , Núcleo Celular/metabolismo , eIF-2 Quinasa/metabolismo , eIF-2 Quinasa/genética , Retículo Endoplásmico/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Lisosomas/metabolismo , Glándulas Mamarias Animales/microbiología , Glándulas Mamarias Animales/metabolismo , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/veterinaria , Infecciones por Mycoplasma/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Transducción de SeñalRESUMEN
Wolfberry is a plant with medicinal and food values. However, its bioactive ingredients and the corresponding genetic bases have not been determined. Here, we de novo generated a chromosome-level genome assembly for wolfberry, yielding a genome sequence of ~1.77 Gb with contig N50 of 50.55 Mb and 39 224 predicted gene models. A variation map, using 307 re-sequenced accessions, was called based on this genome assembly. Furthermore, the fruit metabolome of these accessions was profiled using 563 annotated metabolites, which separated Lycium barbarum L. and non-L. barbarum L. The flavonoids, coumarins, alkaloids and nicotinic acid contents were higher in the former than in the latter. A metabolite-based genome-wide association study mapped 156 164 significant single nucleotide polymorphisms corresponding to 340 metabolites. This included 19 219 unique lead single nucleotide polymorphisms in 1517 significant association loci, of which three metabolites, flavonoids, betaine and spermidine, were highlighted. Two candidate genes, LbUGT (evm.TU.chr07.2692) and LbCHS (evm.TU.chr07.2738), with non-synonymous mutations, were associated with the flavonoids content. LbCHS is a structural gene that interacts with a nearby MYB transcription factor (evm.TU.chr07.2726) both in L. barbarum and L. ruthenicum. Thus, these three genes might be involved in the biosynthesis/metabolism of flavonoids. LbSSADH (evm.TU.chr09.627) was identified as possibly participating in betaine biosynthesis/metabolism. Four lycibarbarspermidines (E-G and O) were identified, and only the lycibarbarspermidines O content was higher in L. barbarum varieties than in non-L. barbarum varieties. The evm.TU.chr07.2680 gene associated with lycibarbarspermidines O was annotated as an acetyl-CoA-benzylalcohol acetyltransferase, suggesting that it is a candidate gene for spermidine biosynthesis. These results provide novel insights into the specific metabolite profile of non-L. barbarum L. and the genetic bases of flavonoids, betaine and spermidine biosynthesis/metabolism.
Asunto(s)
Betaína , Flavonoides , Estudio de Asociación del Genoma Completo , Lycium , Polimorfismo de Nucleótido Simple , Espermidina , Flavonoides/metabolismo , Lycium/genética , Lycium/metabolismo , Espermidina/metabolismo , Betaína/metabolismo , Polimorfismo de Nucleótido Simple/genética , Genoma de Planta/genética , Frutas/genética , Frutas/metabolismoRESUMEN
Carotenoids are important natural pigments and have medical and health functions for humans. Carotenoid cleavage dioxygenase 4 (CCD4) and ethylene responsive factor (ERF) participate in carotenoid metabolism, but their roles in Lycium have not been discovered. Here, we annotated LbCCDs from the Lycium reference genome and found that LbCCD4.1 expression was significantly correlated with the carotenoid metabolites during Lycium five fruit developmental stages. Over-expression of LbCCD4.1 in NQ's leaves resulted in a series of significantly lower contents of carotenoid metabolites, including ß-carotene and ß-cryptoxanthin. Moreover, LbERF5.1, a transcription factor belonging to the ERF family that was located in the nucleus, was isolated. Significant reductions in the carotenoids, especially lutein, violaxanthin and their derivatives, were observed in over-expressing ERF5.1 transgenic NQ's leaves. Over-expression or virus-induced gene silencing of LbERF5.1 in NQ's leaves induced a consistent up- or down-expression, respectively, of LbCCD4.1. Furthermore, yeast one-hybrid and dual-luciferase reporter assays showed that ERF5.1 interacted with the promoter of CCD4.1 to increase its expression, and LbERF5.1 could bind to any one of the three predicted binding sites in the promoter of LbCCD4.1. A transcriptome analysis of LbERF5.1 and LbCCD4.1 over-expressed lines showed similar global transcript expression, and geranylgeranyl diphosphate synthase, phytoene synthase, lycopene δ-cyclase cytochrome, cytochrome P450-type monooxygenase 97A, cytochrome P450-type monooxygenase 97C, and zeaxanthin epoxidase in the carotenoid biosynthesis pathway were differentially expressed. In summary, we uncovered a novel molecular mechanism of carotenoid accumulation that involved an interaction between ERF5.1 and CCD4.1, which may be used to enhance carotenoid in Lycium.
RESUMEN
BACKGROUND: Lycium Linn. (Solanaceae) is a genus of economically important plants producing fruits and leaves with high nutritional value and medicinal benefits. However, genetic analysis of this plant and molecular breeding for quality improvement are limited by the lack of sufficient molecular markers. RESULTS: In this study, two parental strains, 'Ningqi No. 1' (Lycium barbarum L.) and 'Yunnan Gouqi' (Lycium yunnanense Kuang et A.M. Lu), and 200 F1 hybrid individuals were resequenced for genetic analysis. In total, 8,507 well-selected SNPs were developed, and a high-density genetic map (NY map) was constructed with a total genetic distance of 2,122.24 cM. A consensus genetic map was established by integrating the NY map and a previously published genetic map (NC map) containing 15,240 SNPs, with a total genetic distance of 3,058.19 cM and an average map distance of 0.21 cM. The 12 pseudochromosomes of the Lycium reference genome were anchored using this consensus genetic map, with an anchoring rate of 64.3%. Moreover, weak collinearities between the consensus map and the pepper, potato, and tomato genomes were observed. Twenty-five stable QTLs were identified for leaf- and fruit-related phenotypes, including fruit weight, fruit longitude, leaf length, the fruit index, and the leaf index; these stable QTLs were mapped to four different linkage groups, with LOD scores ranging from 2.51 to 19.37 and amounts of phenotypic variance explained from 6.2% to 51.9%. Finally, 82 out of 188 predicted genes underlying stable QTLs for fruit-related traits were differentially expressed according to RNA-seq analysis. CONCLUSIONS: A chromosome-level assembly can provide a foundation for further functional genomics research for wolfberry. The genomic regions of these stably expressed QTLs could be used as targets for further fine mapping and development of molecular markers for marker-assisted selection (MAS). The present study provided valuable information on saturated SNP markers and reliable QTLs for map-based cloning of functional genes related to yield and morphological traits in Lycium spp.
Asunto(s)
Mapeo Cromosómico , Frutas/genética , Ligamiento Genético , Marcadores Genéticos , Lycium/genética , Hojas de la Planta/genética , Sitios de Carácter Cuantitativo , China , Productos Agrícolas/genética , Variación Genética , Fenotipo , Sintenía/genéticaRESUMEN
Hepatocellular carcinoma (HCC) is a frequent malignant tumor. Catalpol is a Chinese medicine extract with a number of pharmacologically active properties. The present study aimed to investigate the effects and mechanisms of catalpol in HCC. HCC cells were treated with catalpol in the presence or absence of microRNA (miR)1405p inhibitor, and assays to determine cell viability, proliferation, invasion and migration were performed. Reverse transcriptionquantitative PCR and western blotting were performed to determine the mRNA and protein expression levels of miR1405p, vimentin, NCadherin and ECadherin. Moreover, cells were treated with catalpol in the absence or presence of transforming growth factor (TGF)ß1, and the cell morphology was observed under a microscope. The results demonstrated that catalpol inhibited cell proliferation, invasion and migration, and decreased the expression levels of vimentin and Ncadherin, but increased the expression levels of Ecadherin and miR1405p. Catalpol inhibited morphological changes in epithelialmesenchymal transformation (EMT) of cells induced by TGFß1. Following inhibition of miR1405p expression, the proliferation, invasion and migration of HCC cells were promoted, Ecadherin expression was decreased, and the levels of vimentin and Ncadherin were increased. The miR1405p inhibitor effectively reversed the inhibitory effect of catalpol on cell proliferation, invasion and migration. Thus, the results suggested that the antitumor potential of catalpol in HCC may be exerted by regulating the expression of miR1405p to inhibit proliferation, invasion, migration and EMT of HCC cells.
Asunto(s)
Carcinoma Hepatocelular/genética , Glucósidos Iridoides/farmacología , Neoplasias Hepáticas/genética , MicroARNs/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Vimentina/genética , Vimentina/metabolismoRESUMEN
Goji berries have been used as food and medicine for millennia. Due to their high morphological similarity, fruits of two distinct species belonging to the family Solanaceae, Lycium barbarum (LB) and Lycium chinense (Chinese boxthorn), are usually marketed together as goji berries, but nearly 90% of all commercially available goji berries belong to the former species. A third closely related species, a wild perennial thorny shrub native to north-western China, Lycium ruthenicum (LR; known as Russian box thorn, and its fruit as black wolfberry), has become a popular choice for combating soil desertification and for alleviating soil salinity/alkalinity due to its high resistance to the harsh environment of saline deserts. Despite the phylogenetic closeness of LB and LR, their fruits are very different. To identify the genes involved in these distinct phenotypes, here we studied expression patterns of 22 transcriptional regulators that may be crucial drivers of these differences during five developmental stages. BAM1 may contribute to higher sugar content in LB. High expression of BFRUCT in ripe LR is likely to be an evolutionary adaptation to fruit ripening in an arid environment. Two arogenate dehydratase paralogues, CHS and LDOX, are probably crucial elements of the mechanism by which LR accumulates much higher levels of anthocyanin. DXS2 (carotenoid accumulation in LB) and CCD4 (carotenoid degradation in ripe LR fruit) may be crucial drivers behind the much higher content of carotenoids in LB. EIL3 and ERF5 are two transcription factors that may contribute to the higher abiotic stress resilience of LR. GATA22-like appears to have more important roles in growth than ripening in LB fruit and vice versa in LR. HAT5-like exhibited opposite temporal patterns in two fruits: high in the 1st stage in LB and high in the 5th stage in LR. PED1 was expressed at a much lower level in LR. Finally, we hypothesise that the poorly functionally characterised SCL32 gene may play a part in the increased resistance to environmental stress of LR. We suggest that BAM1, BFRUCT, EIL3, ERF5, ADT paralogues (for functional redundancy), PED1, GATA22-like, HAT5-like and SCL32 warrant further functional studies.
Asunto(s)
Frutas/metabolismo , Lycium/metabolismo , China , Frutas/genética , Regulación de la Expresión Génica de las Plantas/genética , Lycium/genéticaRESUMEN
Red wolfberry (or goji berry, Lycium barbarum; LB) is an important agricultural product with a high content of pharmacologically important secondary metabolites such as phenylpropanoids. A close relative, black wolfberry (L. ruthenicum; LR), endemic to the salinized deserts of northwestern China, is used only locally. The two fruits exhibit many morphological and phytochemical differences, but genetic mechanisms underlying them remain poorly explored. In order to identify the genes of interest for further studies, we studied transcriptomic (Illumina HiSeq) and metabolomic (LC-MS) profiles of the two fruits during five developmental stages (young to ripe). As expected, we identified much higher numbers of significantly differentially regulated genes (DEGs) than metabolites. The highest numbers were identified in pairwise comparisons including the first stage for both species, but total numbers were consistently somewhat lower for the LR. The number of differentially regulated metabolites in pairwise comparisons of developmental stages varied from 66 (stages 3 vs 4) to 133 (stages 2 vs 5) in both species. We identified a number of genes (e.g. AAT1, metE, pip) and metabolites (e.g. rutin, raffinose, galactinol, trehalose, citrulline and DL-arginine) that may be of interest to future functional studies of stress adaptation in plants. As LB is also highly suitable for combating soil desertification and alleviating soil salinity/alkalinity/pollution, its potential for human use may be much wider than its current, highly localized, relevance.
Asunto(s)
Frutas/genética , Frutas/metabolismo , Lycium/genética , Lycium/metabolismo , Metaboloma , Transcriptoma , Biología Computacional/métodos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Humanos , Redes y Vías Metabólicas , Metabolómica/métodos , Anotación de Secuencia MolecularRESUMEN
Wolfberry (Lycium Linn. 2n = 24) fruit, Gouqizi, is a perennial shrub, traditional food and medicinal plant resource in China. Leaf and fruit related characteristics are economically important traits that are the focus for genetic improvement, but few studies into the molecular genetics of this crop have been reported to date. Here, an F1 population (302 individuals) derived from a cross between "NO.1 Ningqi" (Lycium barbarum L.) and "Chinese gouqi" (Lycium chinese Mill.) was constructed. We recorded fruit weight, longitude, diameter and index along with leaf length, width and index for three consecutive years from 2015 to 2017. Based on this population and these phenotypic data, we constructed the first high-density genetic map of Lycium using specific length amplified fragment sequencing (SLAF-seq) and analyzed quantitative trait loci (QTLs). The map contains 6733 single nucleotide polymorphisms and 12 linkage groups (LG) with a total map distance of 1702.45 cM and an average map distance of 0.253 cM. A total of 55 QTLs were mapped for more than 2 years, of which 18 stable QTLs for fruit index on LG 11, spanning an interval of 73.492-90.945 cM, were detected. qFI11-15 for fruit index was an impressive QTL with logarithm of odds (LOD) and proportion of variance explained (PEV) values reaching 11.07 and 19.7%, respectively. The QTLs on LG 11 were gathered tightly, having an average interval of less than 1 cM per QTL, suggesting that there might be a cluster region controlling fruit index. Remarkably, qLI10-2 and qLI11-2 for leaf index were detectable for 3 years. These results give novel insight into the genetic control of leaf and fruit related traits in Lycium and provide robust support for undertaking further positional cloning studies and implementing marker-assisted selection in seedlings.
RESUMEN
Wolfberry (Lycium barbarum L.) fruits of three cultivars ('Damaye', 'Baihua' and 'Ningqi No.1') were harvested at five different ripening stages and evaluated for sugars and organic acids. Fructose, glucose and total sugar contents increased continually through development and reached their maxima at 34 days after full bloom (DAF). Fructose and glucose were the predominant sugars at maturity, while sucrose content had reduced by maturity. L.barbarum polysaccharides (LBP) content was in the range of 13.03-76.86 mg g(-1)FW during ripening, with a maximum at 20DAF. Citric, tartaric and quinic acids were the main organic acid components during development, and their levels followed similar trends: the highest contents were at 30, 14 and 20DAF, respectively. The significant correlations of fructose and total sugar contents with LBP content during fruit development indicated that they played a key role in LBP accumulation.
Asunto(s)
Carbohidratos/análisis , Ácidos Carboxílicos/análisis , Frutas/química , Lycium/química , Ácido Cítrico/análisis , Fructosa/análisis , Frutas/crecimiento & desarrollo , Glucosa/análisis , Lycium/crecimiento & desarrollo , Polisacáridos/análisis , Ácido Quínico/análisis , Sacarosa/análisis , Tartratos/análisisRESUMEN
RING-finger proteins play crucial roles in ubiquitination events involved in diverse cellular processes including signal transduction, differentiation and apoptosis. Most of the RING-finger proteins have E3-ubiquitin ligase activity. RNF11 is a small RING-finger protein and harbors a RING-H2 domain and a PY motif that could facilitate protein:protein interaction(s) involved in oncogenesis. To isolate RNF11 protein partners and determine its role in normal and cancer cells, we performed yeast two-hybrid screening. Among 18 in-frame positive clones, three were found to be ZBRK1, Eps15 and AMSH (associated molecule with the SH3 domain of STAM). ZBRK1 is a KRAB domain containing Zinc-finger protein and is known to repress target gene transcription in a BRCA1-dependent manner. Eps15 is monoubiquitinated and is part of an essential complex involved in the endocytosis of plasma membrane receptors via the clathrin-mediated internalization pathway. Recent studies have shown that AMSH protein is involved in BMP/TGF-beta signaling pathway by binding to Smad6 and Smad7. The association of RNF11 with these binding partners suggests that it would be involved in biological processes such as gene transcription, BMP/TGF-beta signaling and ubiquitination-associated events. Previously, we have shown that RNF11 interacts with the HECT-type E3 ligases AIP4 and Smurf2. Here, we show that RNF11 binds to AMSH in mammalian cells and that this interaction is independent of the RNF11 RING-finger domain and the PY motif. Our results also demonstrate that AMSH is ubiquitinated by Smurf2 E3 ligase in the presence of RNF11 and that a consequent reduction in its steady-state level requires both RNF11 and Smurf2. RNF11 therefore recruits AMSH to Smurf2 for ubiquitination, leading to its degradation by the 26S proteasome. The potential functions of RNF11-mediated degradation of AMSH in breast cancer are discussed.
Asunto(s)
Proteínas Portadoras/genética , Endopeptidasas , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Sustitución de Aminoácidos , Sitios de Unión , Proteínas Portadoras/metabolismo , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/genética , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Saccharomyces cerevisiae/metabolismo , Transfección , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa , Ubiquitina-Proteína Ligasas/metabolismo , Dedos de Zinc/genética , Dominios Homologos srcRESUMEN
As part of an integrated study of breast cancer gene expression, partial cDNAs were cloned from normal and tumor breast cells by subtractive-hybridization and differential display cloning. The DNA sequence for one of these breast cancer associated genes was used to construct the larger 1319 bp BCA3 cDNA sequence using ESTs without assigned names or functions. High-level BCA3 mRNA expression was found in breast and prostate tumor cell lines whereas normal breast and prostate tissues have low-level expression. Further analysis revealed possible functional domains and alternative splicing of BCA3 that we confirmed by RT-PCR analysis. Immunohistochemistry revealed that the protein is expressed in breast tumor cells in vivo, and not in surrounding stromal tissue.
Asunto(s)
Empalme Alternativo , Neoplasias de la Mama/genética , Proteínas de Neoplasias/genética , Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN Complementario , Etiquetas de Secuencia Expresada , Femenino , Humanos , Datos de Secuencia Molecular , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Especificidad de Órganos , Prolina/metabolismo , Alineación de SecuenciaRESUMEN
CHO 2, encoding human sterol 8-isomerase (hSI), was introduced into plasmids pYX213 or pET23a. The resulting native protein was overexpressed in erg 2 yeast cells and purified to apparent homogeneity. The enzyme exhibited a K (m) of 50 microM and a turnover number of 0.423 s(-1) for zymosterol, an isoelectric point of 7.70, a native molecular mass of 107000 Da and was tetrameric. The structural features of zymosterol provided optimal substrate acceptability. Biomimetic studies of acid-catalysed isomerization of zymosterol resulted in formation of cholest-8(14)-enol, whereas the enzyme-generated product was a Delta(7)-sterol, suggesting absolute stereochemical control of the reaction by hSI. Using (2)H(2)O and either zymosterol or cholesta-7,24-dienol as substrates, the reversibility of the reaction was confirmed by GC-MS of the deuterated products. The positional specific incorporation of deuterium at C-9alpha was established by a combination of (1)H- and (13)C-NMR analyses of the enzyme-generated cholesta-7,24-dienol. Kinetic analyses indicated the reaction equilibrium ( K (eq)=14; DeltaG(o')=-6.5 kJ/mol) for double-bond isomerization favoured the forward direction, Delta(8) to Delta(7). Treatment of hSI with different high-energy intermediate analogues produced the following dissociation constants ( K (i)): emopamil (2 microM)=tamoxifen (1 microM)=tridemorph (1 microM)<25-azacholesterol (21 microM) Asunto(s)
Esteroide Isomerasas/aislamiento & purificación
, Animales
, Secuencia de Bases
, Catálisis
, Cromatografía en Gel
, Cromatografía por Intercambio Iónico
, Cartilla de ADN
, ADN Complementario
, Electroforesis en Gel de Poliacrilamida
, Humanos
, Cinética
, Resonancia Magnética Nuclear Biomolecular
, Proteínas Recombinantes/antagonistas & inhibidores
, Proteínas Recombinantes/química
, Proteínas Recombinantes/genética
, Proteínas Recombinantes/aislamiento & purificación
, Proteínas Recombinantes/metabolismo
, Estereoisomerismo
, Esteroide Isomerasas/antagonistas & inhibidores
, Esteroide Isomerasas/química
, Esteroide Isomerasas/genética
, Esteroide Isomerasas/metabolismo
RESUMEN
Delta(24(28))-Sterols are end products of a mono C-methylation pathway catalyzed by the native Delta(24(25))- to Delta(24(28))-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae. Using a Tyr(81) to Phe mutant SMT enzyme of S. cerevisiae, generated by site-directed mutagenesis of a highly conserved residue in the sterol binding site, we found that several Delta(24(25))- and Delta(24(28))-sterols, which are not substrates for the native protein, were catalyzed to mono- and bis-C24-alkylated side chains. The mutant protein behaved similarly to the native protein in chromatography and in binding zymosterol, the preferred substrate. Zymosterol was converted to fecosterol by the Y81F mutant protein with similar turnover efficiency as the native protein (K(m) = 12 &mgr;M and k(cat) = 0.01 s(-)(1)); trace 24-ethyl sterols were detected from these incubations. 4alpha-Methyl zymosterol, which is not a normal substrate for the wild-type SMT enzyme, was converted to 4alpha-methyl fecosterol in high yield. When fecosterol and 4alpha-methyl fecosterol were assayed individually at saturating concentrations only fecosterol served as an effective substrate for the second C-transfer step (K(m) = 38 &mgr;M and k(cat) = 0.002 s(-)(1)), suggesting that successive C-methylation of Delta(24(28))-substrates is limited by product release and that molecular recognition of sterol features involves hydrogen bond formation. Isomeric 24-ethyl sterol olefins generated from 24(28)-methylene cholesterol were characterized by chromatographic (GC and HPLC) and spectral methods (MS and (1)H NMR), viz., fucosterol, isofucosterol, and clerosterol. Changes in rate of C-methylation and product distributions resulting from deuterium substitution at C28 were used to establish the kinetic isotope effects (KIEs) for the various deprotonations leading to C24-methylene, C24-ethylidene, and C24-ethyl sterols. An isotope effect on C28 methyl deprotonation generated during the first C(1)-transfer was detected with zymosterol and desmosterol paired with AdoMet and [(2)H(3)-methyl]AdoMet. A similar experiment to test for a KIE generated during the second C(1)-transfer reaction with AdoMet paired with 24(28)-methylenecholesterol and [28-(2)H(2)]24(28)-methylene cholesterol indicated an inverse isotope effect associated with C27 deprotonation. Alteration in the proportion of the C24 alkylated olefinic products generated by the pure Y81F mutant resulted from the suppression of the formation of Delta(24(28))-ethylidene sterols (C28 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of 24-ethyl sterols (C27 deprotonation). Kinetic study on the rate of product formation indicated a normal KIE of k(H)/k(D) = 2.62 for the first C(1)-transfer. Alternatively, an inverse KIE was established with k(H)/k(D) = 0.9 for the second C(1)-transfer resulting from conversion of the 24(28)-double bond (sp(2) hybridization) to a 24beta-ethyl group (sp(3) hybridization). From the structures and stereochemical assignments of the C-ethyl olefin products, the stereochemistry of the attack of AdoMet in the second C(1)-transfer was found to operate a Si-face (backside) attack at C24, analogous to the first C(1)-transfer reaction.