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The role of BAHD acyltransferases in poplar (Populus spp.) secondary metabolism and synthesis of salicinoid phenolic glycosidesChedgy, Russell James 24 April 2015 (has links)
The salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae family and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Despite their important role in plant defense, their biosynthetic pathway is not known, although recent work has suggested that benzyl benzoate acts as a possible biosynthetic intermediate. We identified three candidate genes encoding BAHD-type acyltransferases that are predicted to produce benzylated secondary metabolites, named PtACT47, PtACT49, and PtACT54.
Expression of PtACT47 and PtACT49 generally correlated with PG content in a variety of tissues and organs of wild type hybrid poplar plants. This correlation was also found in transgenic hybrid poplar where PG content varied with the level of expression of the condensed tannin regulator MYB134 transcript. In these plants, a suppression of PtACT47 and PtACT49 expression was correlated with lower PG content. In contrast, PtACT54 exhibited very low expression in all tissues tested, and this level of expression was not affected in MYB134 plants.
In order to better understand their possible biochemical functions, cDNA cloning, heterologous expression, and in vitro functional characterization was performed on these three BAHD acyltransferases. Recombinant PtACT47 exhibited a low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM-1 sec-1) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA:salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, specifically benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM-1 sec-1) and lowest Km (55.3 µM) was observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA:benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters at trace levels, for example hexenyl benzoate. Recombinant PtACT54 shares low sequence identity with PtSABT (52.3%) and PtBEBT (52.5%) and exhibited only moderate BEBT-like properties. PtSABT and PtBEBT appear to be paralogs based on their high sequence identity (90.6%) and closely related yet distinct biochemical functions. They likely arose from gene duplication and subsequent functional diversification possibly by neofunctionalization.
Wounding experiments showed that abiotic damage stimulated the synthesis of specific PGs, notably salicin and salicortin within 24-48hrs. This was accompanied by a proportional increase in the expression of PtSABT and PtBEBT. Furthermore, experiments using transgenic RNAi lines with knock-down suppression of PtBEBT, and PtSABT, and both genes simultaneously, provided the first direct evidence that BAHD acyltransferases are important in PG production. PtSABT suppression, both individually and in the double knock-down suppression, significantly lowered salicortin content, particularly in mature leaves. However, a reduced level of PtBEBT expression did not have a significant effect on the PGs measured. This could indicate that BEBT-like activity may be a shared function among closely related BAHDs. The suppression of multiple BEBT-like genes may be necessary to further delineate their functions. / Graduate / rjchedgy@uvic.ca
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Compréhension et prédiction de l'énantiosélectivité des lipases / Comprehension and prediction of lipases enantioselectivityLafaquière, Vincent 19 January 2010 (has links)
Cette étude a porté sur l’analyse de l’énantiosélectivité de la lipase de Burkholderia cepacia (BCL) pour les acides 2-substitués, synthons chiraux d’intérêt pharmaceutique, avec pour objectif d’examiner le rôle de l’accès au site actif enfoui de BCL sur l’énantiosélectivité et de développer une procédure d’ingénierie permettant de créer des mutants d’énantiosélectivité améliorée. Pour traiter le problème, une nouvelle approche de calcul, basée sur des algorithmes de planification de mouvements issus de la robotique a été développée. Elle permet l’exploration conformationnelle des espaces multi-dimensionnels contraints et a été appliquée au calcul des trajectoires de plusieurs racémiques dans le site actif de BCL et à l’identification de résidus pouvant potentiellement gêner le déplacement du substrat le long du site actif. Les résultats obtenus in silico ont révélé une corrélation qualitative avec les valeurs d’énantiosélectivité et ont permis de proposer des cibles de mutagénèse. Sur cette base, l’ingénierie du site actif de BCL a été entreprise pour moduler sélectivement l’accès des énantiomères R et S à la triade catalytique. Un système d’expression hétérologue de BCL chez E. coli compatible avec une expression en microplaque, a été développé. Une librairie de 57 (3x19) mono-mutants sur les positions : Leu17, Val266 et Leu287 a été construite par iPCR puis criblée en utilisant une procédure à moyen débit pour identifier les variants actifs pour l’hydrolyse du pNPB. L’énantiosélectivité de ces mutants a ensuite été évaluée pour l’hydrolyse du racémique (R,S)-2 bromophényl acétate de 2-chloro-éthyle, par utilisation d’une nouvelle procédure de criblage en deep-wells. Ce crible a permis de mettre en évidence plusieurs mutants dont les plus prometteurs ont été caractérisés. Ainsi les mutants Leu17Ser et Leu17Met présentent une augmentation de l’énantiosélectivité d’un facteur 10 accompagnée d’une augmentation de leur activité d’un facteur 4 à 5. Le mutant Val266Gly présente, quant à lui, une inversion de l’énantiosélectivité pour le substrat d’intérêt. L’étude des trajectoires par les techniques de planification combinée à une représentation sous la forme de carte de voxels a été réalisée en parallèle. Pour les mutants sélectionnés, une bonne corrélation a été observée entre les résultats obtenus in silico et expérimentalement. De plus, cela a permis de proposer de nouvelles combinaisons de mutations ayant conduit à l’identification de deux double-mutants Leu17Met/Val266Met et Leu17Ser/Leu287Ile d’énantiosélectivité supérieure à 150 pour le substrat modèle, révélant ainsi l’intérêt de l’approche semi-rationnelle proposée / This work has been focused on the understanding of the Burkholderia cepacia lipase (BCL) enantioselectivity towards 2-substituted acids which are chiral building blocks of pharmaceutical interest. The main objective of this work was the investigation of the potential role of substrate accessibility toward the buried active site of BCL on enantioselectivity and the development of an engineering procedure for the design of enantioselective mutants. To study further this hypothesis, a novel computational approach, based on motion-planning algorithms, originally used in robotics, was developed. It allows the conformational exploration of constrained high-dimensional spaces and was applied to the computation of trajectories for a set of racemates within the catalytic site. This methodology also enables the identification of residues potentially hindering substrates displacement along the active site. Results obtained in silico were correlated qualitatively with experimental values of enantioselectivity. On the basis of these results, engineering of the narrow active site of BCL has been undertaken to modulate selectively the access of R and S enantiomers to the catalytic triade. An heterologous expression system of BCL in E. coli compatible with production at microplate scale was developed. A library of 57 (3x19) variants targeted at positions Leu17, Val266 and Leu287 was built by iPCR and subsequently screened using a medium-throughput procedure to identify active variants against pNPB hydrolysis. Next, the enantioselectivity of these mutants was evaluated towards a given racemate, the (R,S)-2-chloro ethyl 2-bromophenylacetate, using a novel screening procedure developed in deep wells. Such screening enabled the identification of several variants amongst which the most promising were characterized. Mutants Leu17Ser and Leu17Met showed a remarkable 10-fold increase of their enantioselectivity and a 4- and 5-fold improvement of their specific activity. Compared to the wild-type enzyme, mutant Val266Gly displayed a reversed enantioselectivity for the substrate of interest. Investigation of the trajectories using motion-planning techniques combined to a voxel map representation was carried out. For selected variants, a fair correlation was observed between in silico and experimental results. Moreover, this enabled us to suggest novel combinations of mutations that led to the identification of two double-mutants Leu17Met/Val266Met and Leu17Ser/Leu287Ile showing an enantioselectivity value higher than 150 for the racemic substrate, revealing thus the effiency of the semi-rational strategy
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