Loss of YB-1 alleviates liver fibrosis by suppressing epithelial-mesenchymal transition in hepatic progenitor cells.

Previously, we reported that the nuclear translocation of Y-box binding protein 1 (YB-1) is induced by transforming growth factor-ß (TGF-ß) and promotes hepatic progenitor cells (HPCs) expansion. Here, we explored the mechanisms underlying YB-1 translocation and the impact of YB-1 on the epithelial-mesenchymal transition (EMT) in HPCs. YB-1flox/floxcre+/- (YB-1f/fcre+/-) mice and YB-1f/fcre-/- mice were fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or a choline-deficient, ethionine-supplemented (CDE) diet. Liver injury and fibrosis were assessed by performing hematoxylin and eosin (HE) and Masson staining. The expression of collagen and EMT-related markers (E-cadherin, N-cadherin, and Snail) was detected by reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and immunofluorescence analyses. Protein kinase B (AKT) expression in HPCs was silenced via RNA interference. Nuclear YB-1 expression in HPCs was detected via western blotting and immunofluorescence analyses. HPC proliferation was detected by immunofluorescence. Our results indicate that YB-1 transcriptionally regulated the biological behavior of HPCs. HPC-specific YB-1 knockout alleviated liver fibrosis in mice fed with DDC or CDE diet. YB-1 nuclear translocation promoted matrix metallopeptidase 9 transcription. YB-1 depletion in HPCs significantly dampened the EMT and inhibited AKT phosphorylation in vitro and in vivo. AKT knockdown compromised TGF-ß-induced YB-1 nuclear translocation, thereby inhibiting the EMT and HPC proliferation. EMT and AKT were highly activated in HPCs in cirrhotic livers. Collectively, our findings indicate that the loss of YB-1 suppressed EMT in HPCs and alleviated liver fibrosis in mice, and that AKT was essential for TGF-ß-induced YB-1 nuclear translocation and HPC proliferation.

Specific knockdown of Y-box binding protein 1 in hepatic progenitor cells inhibits proliferation and alleviates liver fibrosis.

The proliferation of hepatic progenitor cells (HPCs) contributes to liver regeneration and fibrogenesis during chronic liver injury; however, the mechanism modulating HPC proliferation remains unknown. Y-box binding protein-1 (YB-1) is a transcription factor that regulates the transcription of several genes and is highly expressed in liver injury. We explored the role of YB-1 in HPC proliferation and liver fibrosis. We detected increased expansion of HPCs and elevated levels of YB-1 in HPCs from patients with hepatitis B virus-related fibrosis and choline-deficient ethionine-supplemented or 5-diethoxycarbonyl-1,4-dihydrocollidine diet-induced mice compared with those in control groups. HPC-specific deletion of YB-1 using YB-1flox/flox; Foxl1-Cre+/- mice led to reduced HPC expansion and less collagen deposition in the liver tissues compared with that in Cre-/- mice. In cultured primary HPCs, YB-1 knockdown inhibited HPC proliferation. Further experiments indicated YB-1 negatively regulated p53 expression, and silencing of p53 blocked YB-1 knockdown-mediated inhibition of HPC proliferation. Collectively, YB-1 negatively regulates HPC proliferation and alleviates liver fibrosis by p53.

S-Adenosyl-l-ethionine is a Catalytically Competent Analog of S-Adenosyl-l-methione (SAM) in the Radical SAM Enzyme HydG.

Radical S-adenosyl-l-methionine (SAM) enzymes initiate biological radical reactions with the 5'-deoxyadenosyl radical (5'-dAdo•). A [4Fe-4S]+ cluster reductively cleaves SAM to form the Ω organometallic intermediate in which the 5'-deoxyadenosyl moiety is directly bound to the unique iron of the [4Fe-4S] cluster, with subsequent liberation of 5'-dAdo•. Here we present synthesis of the SAM analog S-adenosyl-l-ethionine (SAE) and show SAE is a mechanistically-equivalent SAM-alternative for HydG, both supporting enzymatic turnover of substrate tyrosine and forming the organometallic intermediate Ω. Photolysis of SAE bound to HydG forms an ethyl radical trapped in the active site. The ethyl radical withstands prolonged storage at 77 K and its EPR signal is only partially lost upon annealing at 100 K, making it significantly less reactive than the methyl radical formed by SAM photolysis. Upon annealing above 77K, the ethyl radical adds to the [4Fe-4S]2+ cluster, generating an ethyl-[4Fe-4S]3+ organometallic species termed ΩE.

Identification of an Important Odorant Precursor in Durian: First Evidence of Ethionine in Plants.

On the basis of the following data from the literature, we hypothesized the presence of ethionine in durian pulp: (1) the major odorants in terms of quantity as well as odor potency in durian pulp are ethanethiol and its derivatives; (2) genome analysis of durian assigned methionine γ-lyase (MGL), the enzyme that converts methionine to methanethiol, a key role for durian odor formation; and (3) MGL accepts not only methionine but also ethionine as a substrate. A targeted search by liquid chromatography-tandem mass spectrometry allowed us to confirm the presence of ethionine in durian pulp. Quantitation of ethionine in samples of different varieties (Monthong, Krathum, Chanee, and Kanyao) showed concentrations (621-9600 µg/kg) in the same range but below the methionine concentrations (16100-30200 µg/kg). During fruit ripening, the ethionine concentration increased as well as the ethanethiol concentration. Final evidence for the role of ethionine as an ethanethiol precursor was provided by demonstrating the formation of (2H5)ethanethiol after adding (2H5)ethionine to durian pulp.

Two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Colonization of the gut by certain probiotic Lactobacillus reuteri strains has been associated with reduced risk of inflammatory diseases and colorectal cancer. Previous studies pointed to a functional link between immunomodulation, histamine production, and folate metabolism, the central 1-carbon pathway for the transfer of methyl groups. Using mass spectrometry and NMR spectroscopy, we analyzed folate metabolites of L. reuteri strain 6475 and discovered that the bacterium produces a 2-carbon-transporting folate in the form of 5,10-ethenyl-tetrahydrofolyl polyglutamate. Isotopic labeling permitted us to trace the source of the 2-carbon unit back to acetate of the culture medium. We show that the 2C folate cycle of L. reuteri is capable of transferring 2 carbon atoms to homocysteine to generate the unconventional amino acid ethionine, a known immunomodulator. When we treated monocytic THP-1 cells with ethionine, their transcription of TNF-α was inhibited and cell proliferation reduced. Mass spectrometry of THP-1 histones revealed incorporation of ethionine instead of methionine into proteins, a reduction of histone-methylation, and ethylation of histone lysine residues. Our findings suggest that the microbiome can expose the host to ethionine through a novel 2-carbon transporting variant of the folate cycle and modify human chromatin via ethylation.-Röth, D., Chiang, A. J., Hu, W., Gugiu, G. B., Morra, C. N., Versalovic, J., Kalkum, M. The two-carbon folate cycle of commensal Lactobacillus reuteri 6475 gives rise to immunomodulatory ethionine, a source for histone ethylation.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Intriguing cellular processing of a fluorinated amino acid during protein biosynthesis in Escherichia coli.

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.

Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases.

Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.

The role of surface electrostatics on the stability, function and regulation of human cystathionine ß-synthase, a complex multidomain and oligomeric protein.

Human cystathionine ß-synthase (hCBS) is a key enzyme of sulfur amino acid metabolism, controlling the commitment of homocysteine to the transsulfuration pathway and antioxidant defense. Mutations in hCBS cause inherited homocystinuria (HCU), a rare inborn error of metabolism characterized by accumulation of toxic homocysteine in blood and urine. hCBS is a complex multidomain and oligomeric protein whose activity and stability are independently regulated by the binding of S-adenosyl-methionine (SAM) to two different types of sites at its C-terminal regulatory domain. Here we study the role of surface electrostatics on the complex regulation and stability of hCBS using biophysical and biochemical procedures. We show that the kinetic stability of the catalytic and regulatory domains is significantly affected by the modulation of surface electrostatics through noticeable structural and energetic changes along their denaturation pathways. We also show that surface electrostatics strongly affect SAM binding properties to those sites responsible for either enzyme activation or kinetic stabilization. Our results provide new insight into the regulation of hCBS activity and stability in vivo with implications for understanding HCU as a conformational disease. We also lend experimental support to the role of electrostatic interactions in the recently proposed binding modes of SAM leading to hCBS activation and kinetic stabilization.

Characterization of isolated yeast growth response to methionine analogs.

Methionine is one of the first limiting amino acids in poultry nutrition. The use of methionine-rich natural feed ingredients, such as soybean meal or rapeseed meal may lead to negative environmental consequences. Amino acid supplementation leads to reduced use of protein-rich ingredients. The objectives of this study were isolation of potentially high content methionine-containing yeasts, quantification of methionine content in yeasts and their respective growth response to methionine analogs. Minimal medium was used as the selection medium and the isolation medium of methionine-producing yeasts from yeast collection and environmental samples, respectively. Two yeasts previously collected along with six additional strains isolated from Caucasian kefir grains, air-trapped, cantaloupe, and three soil samples could grow on minimal medium. Only two of the newly isolated strains, K1 and C1, grew in minimal medium supplied with either methionine analogs ethionine or norleucine at 0.5% (w/v). Based on large subunit rRNA sequences, these isolated strains were identified as Pichia udriavzevii/Issatchenkia orientalis. P. kudriavzevii/I. orentalis is a generally recognized as a safe organism. In addition, methionine produced by K1 and C1 yeast hydrolysate yielded 1.3 ± 0.01 and 1.1 ± 0.01 mg g(-1) dry cell. Yeast strain K1 may be suitable as a potential source of methionine for dietary supplements in organic poultry feed but may require growth conditions to further increase their methionine content.

The association between progression of atherosclerosis and the methylated amino acids asymmetric dimethylarginine and trimethyllysine.

OBJECTIVE: We previously showed that treatment with folic acid (FA)/B12 was associated with more rapid progression of coronary artery disease (CAD). High doses of FA may induce methylation by increasing the availability of S-adenosyl-methionine (SAM). Asymmetric dimethylarginine (ADMA) and trimethyllysine (TML) are both produced through proteolytic release following post-translational SAM-dependent methylation of precursor amino acid. ADMA has previously been associated with CAD. We investigated if plasma levels of ADMA and TML were associated with progression of CAD as measured by quantitative coronary angiography (QCA). METHODS: 183 patients from the Western Norway B Vitamin Intervention Trial (WENBIT) undergoing percutaneous coronary intervention (PCI) were randomized to daily treatment with 0.8 mg FA/0.4 mg B12 with and without 40 mg B6, B6 alone or placebo. Coronary angiograms and plasma samples of ADMA and TML were obtained at both baseline and follow-up (median 10.5 months). The primary end-point was progression of CAD as measured by diameter stenosis (DS) evaluated by linear quantile mixed models. RESULTS: A total of 309 coronary lesions not treated with PCI were identified. At follow-up median (95% CI) DS increased by 18.35 (5.22-31.49) percentage points per µmol/L ADMA increase (p-value 0.006) and 2.47 (0.37-4.58) percentage points per µmol/L TML increase (p-value 0.021) in multivariate modeling. Treatment with FA/B12 (±B6) was not associated with ADMA or TML levels. CONCLUSION: In patients with established CAD, baseline ADMA and TML was associated with angiographic progression of CAD. However, neither ADMA nor TML levels were altered by treatment with FA/B12 (±B6). TRIAL REGISTRATION: Controlled-Trials.com NCT00354081.

L-leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst.

L-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of L-leucine and L-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of L-methionine and L-ethionine in the same manner as previously described L-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

Small-molecule inhibitors of the protein methyltransferase SET7/9 identified in a high-throughput screen.

Aberrant expression of chromatin-modifying enzymes (CMEs) is associated with a range of human diseases, including cancer. CMEs are now an important target area in drug discovery. Although the role that histone and protein (lysine) methyltransferases (PMTs) play in the regulation of transcription and cell growth is increasingly recognized, few small-molecule inhibitors of this class of enzyme have been reported. Here we describe an assay suitable for primary compound screening for the identification of PMT inhibitors. The assay followed the methylation of histones in the presence of the PMT SET7/9 and the radioactive cofactor S-adenosyl-methionine using scintillating microplates (FlashPlate) and was used to screen approximately 65 000 compounds (% coefficient of variation = 10%; Z' = 0.6). The hits identified from a library of more than 63 000 diverse small molecules included a series of rhodanine compounds with micromolar activity. A screen of the National Cancer Institute Diversity Set (2000 compounds) identified an orsein derivative that inhibited SET7/9 (~20 µM) and showed modest growth inhibition associated with the expected cellular phenotype of reduced histone methylation in a human tumor cell line. The assay represents a useful tool for the identification of inhibitors of PMT activity.

Automated annotation and quantification of metabolites in 1H NMR data of biological origin.

In (1)H NMR metabolomic datasets, there are often over a thousand peaks per spectrum, many of which change position drastically between samples. Automatic alignment, annotation, and quantification of all the metabolites of interest in such datasets have not been feasible. In this work we propose a fully automated annotation and quantification procedure which requires annotation of metabolites only in a single spectrum. The reference database built from that single spectrum can be used for any number of (1)H NMR datasets with a similar matrix. The procedure is based on the generalized fuzzy Hough transform (GFHT) for alignment and on Principal-components analysis (PCA) for peak selection and quantification. We show that we can establish quantities of 21 metabolites in several (1)H NMR datasets and that the procedure is extendable to include any number of metabolites that can be identified in a single spectrum. The procedure speeds up the quantification of previously known metabolites and also returns a table containing the intensities and locations of all the peaks that were found and aligned but not assigned to a known metabolite. This enables both biopattern analysis of known metabolites and data mining for new potential biomarkers among the unknowns.

Identification and characterization of genes related to the production of organic acids in yeast.

Organic acids contribute to the flavor of many foods and drinks including alcoholic beverages. To study the cellular processes affecting organic acid production, here we screened collections of Saccharomyces cerevisiae deletion mutants and identified 36 yeast mutants forming a yellow halo on YPD plates containing bromocresol purple, indicating that the pH of the medium had been lowered. The disrupted genes encoded TCA cycle enzymes, transcription factors, signal transducers, and ubiquitin-related proteins. Acetate, pyruvate, and succinate are produced by yeast fermentation in rich medium, and their production was affected by mutations of the genes GTR1, GTR2, LIP5, LSM1, PHO85, PLM2, RTG1, RTG2 and UBP3, and also succinate dehydrogenase-related genes including EMI5, SDH1, SDH2, SDH4, TCM62 and YDR379C-A. Among the genes identified, overexpression of only LIP5 affected the production of acetate in S. cerevisiae. However, overexpression of EMI5, LIP5, RTG2 and UBP3 had a significant effect on the production of acetate, citrate, lactate, and succinate in the bottom-fermenting yeast Saccharomyces pastorianus. Furthermore, phenotypic analysis of the S. cerevisiae disruptants involved in organic acid production showed that azaserine, citrate, ethionine, and sulfite are useful compounds by which mutants with altered organic acid production might be selected. Taken together, these results suggest that the regulation of many organic acids might be simultaneously achieved by activation or inactivation of a single gene.

Imprinting: RNA expression for homocysteine recycling in the human oocyte.

OBJECTIVE: To investigate whether homocysteine, a well known inhibitor of methylation, which is produced after imprinting and other methylation processes, can be recycled to methionine in the oocyte, at least until the stage of maternal to zygotic transition (i.e., four- to eight-cell stage); before this stage, most of the biochemical processes are carried out with the use of maternal stores of protein and mRNA. DESIGN: A first approach using microarrays and then reverse-transcription polymerase chain reaction (RT-PCR) for methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase [MTR]), betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta synthase (CBS). SETTING: Two private hospitals. PATIENT(S): Patients involved in IVF/ICSI procedures. INTERVENTION(S): Germinal vesicle oocytes collected at the time of oocyte retrieval, RNA extraction amplification, RT-PCR, microarrays. MAIN OUTCOME MEASURE(S): mRNA expression of all the enzymes involved in the chain of methylation and recycling of homocysteine to methionine. RESULT(S): All of the enzymes required for methylation are present in the oocyte. Homocysteine can be recycled with BHMT and MTR. CONCLUSION(S): The human oocyte is able to regulate its Hcy level via remethylation using MTR and BHMT but not CBS. This aspect is important, because recent studies have shown that controlled ovarian hyperstimulation affects the homocysteine concentration in follicular fluid. This may regulate, at least in part, the risk of imprinting problems during IVF procedures.

X-ray structure of the [FeFe]-hydrogenase maturase HydE from Thermotoga maritima.

Maturation of the [FeFe]-hydrogenase active site depends on at least the expression of three gene products called HydE, HydF, and HydG. We have solved the high resolution structure of recombinant, reconstituted S-adenosine-L-methionine-dependent HydE from Thermotoga maritima. Besides the conserved [Fe(4)S(4)] cluster involved in the radical-based reaction, this HydE was reported to have a second [Fe(4)S(4)] cluster coordinated by three Cys residues. However, in our crystals, depending on the reconstitution and soaking conditions, this second cluster is either a [Fe(2)S(2)] center, with water occupying the fourth ligand site or is absent. We have carried out site-directed mutagenesis studies on the related HydE from Clostridium acetobutylicum, along with in silico docking and crystal soaking experiments, to define the active site region and three anion-binding sites inside a large, positive cavity, one of which binds SCN(-) with high affinity. Although the overall triose-phosphate isomerase-barrel structure of HydE is very similar to that of biotin synthase, the residues that line the internal cavity are significantly different in the two enzymes.

Misincorporation of amino acid analogues into proteins by biosynthesis.

Despite astounding diversity in their structure and function, proteins are constructed from 22 protein or 'canonical' amino acids. Hundreds of amino acid analogues exist; many occur naturally in plants, some are synthetically produced or can be produced in vivo by oxidation of amino acid side-chains. Certain structural analogues of the protein amino acids can escape detection by the cellular machinery for protein synthesis and become misincorporated into the growing polypeptide chain of proteins to generate non-native proteins. In this review we seek to provide a comprehensive overview of the current knowledge on the biosynthetic incorporation of amino acid analogues into proteins by mammalian cells. We highlight factors influencing their incorporation and how the non-native proteins generated can alter cell function. We examine the ability of amino acid analogues, representing those commonly found in damaged proteins in pathological tissues, to be misincorporated into proteins by cells in vitro, providing us with a useful tool in the laboratory to generate modified proteins representing those present in a wide-range of pathologies. We also discuss the evidence for amino acid analogue incorporation in vivo and its association with autoimmune symptoms. We confine the review to studies in which the synthetic machinery of cell has not been modified to accept non-protein amino acids.

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum.

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Isolation, cloning and expression of a multifunctional O-methyltransferase capable of forming 2,5-dimethyl-4-methoxy-3(2H)-furanone, one of the key aroma compounds in strawberry fruits.

Strawberry fruits contain an uncommon group of key aroma compounds with a 2,5-dimethyl-3(2H)-furanone structure. Here, we report on the methylation of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) by a S-adenosyl-L-methionine dependent O-methyltransferase, the cloning of the corresponding cDNA and characterization of the encoded protein. Northern-hybridization indicated that the Strawberry-OMT specific transcripts accumulated during ripening in strawberry fruits and were absent in root, petiole, leaf and flower. The protein was functionally expressed in E. coli and exhibited a substrate specificity for catechol, caffeic acid, protocatechuic aldehyde, caffeoyl CoA and DMHF. A common structural feature of the accepted substrates was a o-diphenolic structure also present in DMHF in its dienolic tautomer. FaOMT is active as a homodimer and the native enzyme shows optimum activity at pH 8.5 and 37 degrees C. It does not require a cofactor for enzymatic activity. Due to the expression pattern of FaOMT and the enzymatic activity in the different stages of fruit ripening we suppose that FaOMT is involved in lignification of the achenes and the vascular bundles in the expanding fruit. In addition, it is concluded that the Strawberry-OMT plays an important role in the biosynthesis of strawberry volatiles such as vanillin and DMMF.