Enzymatic reduction of phenylglyoxal and 2,3-butanedione, two commonly used arginine-modifying reagents, by the ketoacyl reductase domain of fatty acid synthase

Fatty acid synthase catalyzes the reduction of one of the carbonyl groups in phenylglyoxal and 2,3-butanedione using NADPH as the reductant. Selective inactivation of the enoyl reductase, one of the two reductase domains that could catalyze this reduction, did not affect the carbonyl reduction showing that the ketoreductase domain catalyzed the reaction. The apparent Km for the two arginine-specific reagents were lower than that for 3-acetoacetyl-N-acetyl cysteamine, the commonly used model substrate for the ketoreductase activity of the synthase.     

Targeting viral infection by microRNA inhibition

An inhibitor of microRNA-122 reduces viral load in chimpanzees that are chronically infected with hepatitis C virus, suggesting that such an approach might have therapeutic potential in humans.     

Biosynthesis of pentahydroxystearic acid of cutin from linoleic acid in Rosmarinus officinalis

The hydroxyfatty acid polymer, cutin, is the structural component of plant cuticle. Combined gas chromatography-mass spectrometry of the hydrogenolysis and deuterolysis products of rosemary cutin (Rosmarinus officinalis) revealed a series of components suggesting the conversion of linoleic acid to 9,10,12,13,18-pentahydroxy-stearic acid. [U-¹⁴C]Linoleic acid was incorporated into the insoluble residue of rapidly expanding rosemary leaves. Depolymerization of the insoluble material followed by isolation of individual components and chemical degradation studies showed that linoleic acid was directly converted into 18-hydroxylinoleic acid, 18-hydroxy-9, 10-epoxyoctadec-12-enoic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, 9,10,18-trihydroxy-12,13-epoxystearic acid, and 9,10,12,13,18-pentahydroxystearic acid. These results strongly suggest that, in the biosynthesis of the phytopolymer, linoleic acid is first converted into 18-hydroxylinoleic acid and that this precursor then undergoes sequential epoxidation-hydration at the Δ⁹ and Δ¹² double bonds to yield 9,10,12,13,18-pentahydroxystearic acid.     

Fruit-specific overexpression of wound-induced tap1 under E8 promoter in tomato confers resistance to fungal pathogens at ripening stage

Based on high economic importance and nutritious value of tomato fruits and as previous studies employed E8 promoter in fruit ripening-specific gene expression, we have developed transgenic tomato plants overexpressing tomato anionic peroxidase cDNA (tap1) under E8 promoter. Stable transgene integration was confirmed by polymerase chain reaction (PCR) and Southern analysis for nptII. Northern blotting confirmed elevated tap1 levels in the breaker- and red-ripe stages of T(1) transgenic fruits, whereas wild-type (WT) plants did not show tap1 expression in these developmental stages. Further, tap1 expression levels were significantly enhanced in response to wounding in breaker- and red-ripe stages of transgenic fruits, whereas wound-induced expression of tap1 was not detected in WT fruits. Confocal microscopy revealed high accumulation of phenolic compounds at the wound site in transgenic fruits suggesting a role of tap1 in wound-induced phenolic polymerization. Total peroxidase activity has increased remarkably in transgenic pericarp tissues in response to wounding, while very less or minimal levels were recorded in WT pericarp tissues. Transgenic fruits also displayed reduced post-harvest decay and increased resistance toward Alternaria alternata and Fusarium solani infection with noticeable inhibition in lesion formation. Conidiospore germination and mycelial growth of F. solani were severely inhibited when treated with E8-tap1 fruit extracts compared to WT fruits. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay showed reduced spore viability when incubated in E8-tap1 fruit extracts. Thus, fruit-specific expression of tap1 using E8 promoter is associated with enhanced total peroxidase activity and high phenolic accumulation in fruits with minimized post-harvest deterioration caused by wounding and fungal attack in tomato fruits.     

Identification and characterization of Rv3281 as a novel subunit of a biotin-dependent acyl-CoA Carboxylase in Mycobacterium tuberculosis H37Rv

Mycobacterium tuberculosis produces a large number of structurally diverse lipids generated from the carboxylation products of acetyl-CoA and propionyl-CoA. A biotin-dependent acyl-CoA carboxylase was purified from M. tuberculosis H37Rv by avidin affinity chromatography, and the three major protein components were determined by N-terminal sequencing to be the 63-kDa alpha3-subunit (AccA3, Rv3285), the 59-kDa beta5-subunit (AccD5, Rv3280), and the 56-kDa beta4-subunit (AccD4, Rv3799). A minor protein of about 24 kDa that co-purified with the above subunits was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to be the product of Rv3281 that is located immediately downstream of the open reading frame encoding the beta5-subunit. This protein displays identity over a short stretch of amino acids with the recently discovered epsilon-subunits of Streptomyces coelicolor, suggesting that it might be an epsilon-subunit of the mycobacterial acyl-CoA carboxylase. To test this hypothesis, the carboxylase subunits were expressed in Escherichia coli and purified. Acyl-CoA carboxylase activity was successfully reconstituted for the first time from purified subunits of the acyl-CoA carboxylase of M. tuberculosis. The reconstituted alpha3-beta5 showed higher activity with propionyl-CoA than with acetyl-CoA, and the addition of the epsilon-subunit stimulated the carboxylation by 3.2- and 6.3-fold, respectively. The alpha3-beta4 showed very low activity with the above substrates but carboxylated long chain acyl-CoA. This epsilon-subunit contains five sets of tandem repeats at the N terminus that are required for maximal enhancement of carboxylase activity. The Rv3281 open reading frame is co-transcribed with Rv3280 in the mycobacterial cell, and the level of epsilon-protein was highest during the log phase and decreased during the stationary phase.     

Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells

Lipids that are uniquely found in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched long-chain fatty acids, have long been thought to play a role in host-pathogen interactions. The recent construction by Dubey et al. (2002) Mol Microbiol 45: 1451-1459, of a Mycobacterium tuberculosis mutant that is deficient in the synthesis of the di- and tri-methylbranched fatty acids, mycolipenates and mycosanoates, found in some forms of diacyltrehaloses (DAT) and polyacyltrehaloses (PAT) provided the opportunity to assess the contribution of these complex lipids to pathogenesis directly. We provide evidence that DAT/PAT deficiency affects the surface global composition of the mycobacterial cell envelope improving the efficiency with which M. tuberculosis binds to and enters phagocytic and non-phagocytic host cells. Interestingly, this property did not affect the overall replication and persistence of the tubercle bacillus in the lungs, spleen and liver of mice infected via the respiratory or intravenous route.     

Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase,a novel fungal virulence factor

Hard surface contact has been known to be necessary to induce infection structure (appressorium) formation in many phytopathogenic fungi. However, the molecular basis of this requirement is unknown. We have used a differential display approach to clone some of the genes induced in the conidia by hard surface contact. We report that one of the genes induced by hard-surface contact of the conidia of Colletotrichum gloeosporioides, chip6, encodes a protein with homology to sterol glycosyl transferases. chip6 expressed in E. coli catalyses glucosyl transfer from UDP-glucose to cholesterol. Disruption of chip6 causes a marked decrease in the transferase activity and a drastic reduction in virulence on its natural host, avocado fruits, although the mutant is capable of normal growth and appressorium formation. The requirement for sterol glycosyl transferase for pathogenicity suggests a novel biological function for this transferase.