Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.     

Regulation of monoterpene accumulation in leaves of peppermint

Plants synthesize numerous classes of natural products that accumulate during development and are thought to function as constitutive defenses against herbivores and pathogens. However, little information is available about how the levels of such defenses are regulated. We measured the accumulation of monoterpenes, a model group of constitutive defenses, in peppermint (Mentha x piperita L.) leaves and investigated several physiological processes that could regulate their accumulation: the rate of biosynthesis, the rate of metabolic loss, and the rate of volatilization. Monoterpene accumulation was found to be restricted to leaves of 12 to 20 d of age, the period of maximal leaf expansion. The rate of monoterpene biosynthesis determined by (14)CO(2) incorporation was closely correlated with monoterpene accumulation, as determined by gas chromatographic analysis, and appeared to be the principal factor controlling the monoterpene level of peppermint leaves. No significant catabolic losses of monoterpenes were detected throughout leaf development, and monoterpene volatilization was found to occur at a very low rate, which, on a monthly basis, represented less than 1% of the total pool of stored monoterpenes. The composition of volatilized monoterpenes differed significantly from that of the total plant monoterpene pool, suggesting that these volatilized products may arise from a separate secretory system. With the demonstration that the rate of biosynthesis is the chief process that determines monoterpene accumulation in peppermint, efforts to improve production in this species can now focus on the genes, enzymes, and cell differentiation processes that regulate monoterpene biosynthesis.     

'Close-fitting sleeves': DNA damage recognition by the UvrABC nuclease system

DNA damage recognition represents a long-standing problem in the field of protein-DNA interactions. This article reviews our current knowledge of how damage recognition is achieved in bacterial nucleotide excision repair through the concerted action of the UvrA, UvrB, and UvrC proteins.     

Molecular cloning of a taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli

The taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-induced Taxus cells, and partially purified and characterized (K. Walker, R. E. B. Ketchum, M. Hezari, D. Gatfield, M. Golenowski, A. Barthol, and R. Croteau, Arch. Biochem. Biophys. 364, 273-279 1999). A revised purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment. This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)(+) RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence was obtained. Expression of this clone from pCWori(+) in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verification of the acetylated product. The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information. Sequence comparison of the taxadien-5alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64-67%). The efficient conversion of taxadien-5alpha-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production.     

Breastfeeding Initiation: Impact of Obesity in a Large Canadian Perinatal Cohort Study

ObjectiveTo evaluate incidence of breastfeeding initiation according to maternal pre-pregnancy body mass index (BMI) in “Grossesse en Santé”, a large prospective birth cohort in Quebec City.MethodsBreastfeeding initiation in the post-partum period, pre-pregnancy BMI, sociodemographic determinants and obstetrical and neonatal factors were collected from years 2005 to 2010 in 6592 women with single pregnancies. Prenatal non-intention to breastfeed was documented in a subgroup of the cohort (years 2009–2010). Log-binomial regression analyses were performed to assess relative risk (RR) of non-initiation of breastfeeding between maternal BMI categories in models including pre- and post-natal determinants, after exclusion of variables with a mediating effect.ResultsTwenty percent (20%) of obese women did not initiate breastfeeding in the post-natal period at hospital compared to 12% for normal weight women. Compared with those having a normal pre-pregnancy BMI, obese women had a higher risk of non-initiation of breastfeeding (RRunadj 1.69, 95% CI 1.44–1.98), even after adjustment for prenatal and sociodemographic factors (RRadj 1.26, 95% CI 1.08–1.46). Furthermore, the risk of non-initiation of breastfeeding in obese women still remained higher after introduction of per- and post-natal factors (RR 1.22, 95% CI 1.04–1.42). The prenatal non-intention to breastfeed was strongly associated with the non-initiation of breastfeeding for all categories of BMI.ConclusionMaternal obesity is associated with a two-fold rate of non-initiation of breastfeeding. Considering the benefits of breastfeeding and the increasing obesity rate, adapted interventions and specialized support should target both pre- and immediate post-natal periods in this population.     

Functional deficit associated with a missense Werner syndrome mutation

Werner syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WRN gene. WRN helicase, a member of the RecQ helicase family, is involved in various DNA metabolic pathways including DNA replication, recombination, DNA repair and telomere maintenance. In this study, we have characterized the G574R missense mutation, which was recently identified in a WS patient. Our biochemical experiments with purified mutant recombinant WRN protein showed that the G574R mutation inhibits ATP binding, and thereby leads to significant decrease in helicase activity. Exonuclease activity of the mutant protein was not significantly affected, whereas its single strand DNA annealing activity was higher than that of wild type. Deficiency in the helicase activity of the mutant may cause defects in replication and other DNA metabolic processes, which in turn could be responsible for the Werner syndrome phenotype in the patient. In contrast to the usual appearance of WS, the G574R patient has normal stature. Thus the short stature normally associated with WS may not be due to helicase deficiency.     

An assessment of gene‐by‐environment interactions in developmental dyslexia‐related phenotypes

While the genetic and environmental contributions to developmental dyslexia (DD) have been studied extensively, the effects of identified genetic risk susceptibility and of specified environmental hazardous factors have usually been investigated separately. We assessed potential gene‐by‐environment (GxE) interactions on DD‐related reading, spelling and memory phenotypes. The presence of GxE effects were investigated for the DYX1C1, DCDC2, KIAA0319 and ROBO1 genes, and for seven specified environmental moderators in 165 nuclear families in which at least one member had DD, by implementing a general test for GxE interaction in sib‐pair‐based association analysis of quantitative traits. Our results support a diathesis‐stress model for both reading and memory composites: GxE effects were found between some specified environmental moderators (i.e. maternal smoke during pregnancy, birth weight and socio‐economic status) and the DYX1C1‐1259C/G marker. We have provided initial evidence that the joint analysis of identified genetic risk susceptibility and measured putative risk factors can be exploited in the study of the etiology of DD and reading‐related neuropsychological phenotypes, and may assist in identifying/preventing the occurrence of DD.