Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

Mirroring Enzymes: The Role of H-Bonding In HQuin-BAM Catalyzed Asymmetric Aza-Henry Reactions: A DFT Study into the Reactivity, Mechanism, and Origins of Selectivity

The exact mechanistic understanding of various organocatalytic systems in asymmetric reactions such as Henry and aza-Henry transformations is important for developing and designing new synthetic organocatalysts. The focus of this dissertation will be on the use of density functional theory (DFT) for studying the asymmetric aza-Henry reaction. The first part of the thesis is a detailed mechanistic investigation of a poorly understood chiral bis(amidine) (BAM) Brønsted acid catalyzed aza-Henry reaction between nitromethane and N-Boc phenylaldimine. The catalyst, in addition to acting as a Brønsted base, serves to simultaneously activate both the electrophile and the nucleophile through dual H-bonding during C-C bond formation and is thus essential for both reaction rate and selectivity. Analysis of the H-bonding interactions revealed that there was a strong preference for the formation of a homonuclear positive charge-assisted H-bond, which in turn governed the relative orientation of substrate binding. Attracted by this well-defined mechanistic investigation, the other important aspect of my PhD research addressed a detailed theoretical analysis accounting for the observed selectivity in diastereoselective versions of this reaction. A detailed inspection of the stereodetermining C-C bond forming transition states for monoalkylated nitronate addition to a range of electronically different aldimines, revealed that the origins of stereoselectivity were controlled by a delicate balance of different factors such as steric, orbital interactions, and the extent of distortion in the catalyst and substrates. The structural analysis of different substituted transition states established an interesting dependency on matching the shape and size of the catalyst (host molecule) and substrates (guest molecules) upon binding, both being key factors governing selectivity, in essence, offering an analogy to positive cooperative binding effect of catalytic enzymes and substrates in Nature. In addition, both intra-molecular (intra-host) and inter-molecular (host-guest, guest-guest) stabilizing interactions play a key role to the high π-facial selectivity. The application of dispersion-corrected functionals (i.e., ωB97X-D and B3LYP-D3) was essential for accurately modeling these stabilizing interactions, indicating the importance of dispersion effects in enantioselectivity. As a brief prelude to more extensive future studies, the influence of a triflate counterion on both reactivity and selectivity in this reaction was also addressed.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

A comparative study of protoheme and heme d catalases : role of the heme and the heme pocket in catalysis and ligand binding

Catalase dismutes H20 2 to O2 and H20. In successive twoelectron reactions H20 2 induces both oxidation and reduction at the heme group. In the first step the protoheme prosthetic group of beef liver catalase forms compound I, in which the heme has been oxidized from Fe3+ to Fe4+=0 and a porphyrin radical has been created. Compound II is formed by the oneelectron reduction of comp I. It retains Fe4+=0 but lacks the porphyrin radical and is catalytically inert. Molecular structures are available for Escherichia coli Hydroperoxidase II, Micrococcus Iysodeiktus, Penicillium vitale and beef liver enzymes, which contain different hemes and heme pockets. In the present work, the pockets and substrate access channels of protoheme (beef liver & Micrococcus) and heme d (HPII of E. coli and Penicillium) catalases have been analysed using Quanta™ and CharmMTM molecular modeling packages on the Silicon Graphics Iris Indigo 2 computer. Experimental studies have been carried out with two catalases, HPII (and its mutants) and beef liver. Fluoride and formate' are inhibitors of both enzymes, and their binding is modulated by the heme and by distal residues N201 & H128. Both HPII and beef liver enzymes form compound I with H202 or peracetate. The reduction of beef liver enzyme compound I to II and the decay of compound II are accelerated by fluoride. The decay of compound II is also accelerated by formate, and this reagent acts as a 2-electron donor towards compound I of both enzymes. It is concluded that heme d enzymes (Penicillium and HPII of E. coli) are formed by autocatalytic transformation of protoheme in a modified pocket which contains a characteristic serine residue as well as a partially occluded heme channel. They are less active than protoheme enzymes but also do not form the inactive compound II species. Binding of peroxide as well as fluoride and formate is prevented by mutation of H128 and modulated by mutation of N201.

Identification and characterization of a Catharanthus roseus mutant altered in monoterpenoid indole alkaloid biosynthesis

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source for several pharmaceutically valuable monoterpenoid indole alkaloids (MIAs), including the powerful antihypertensive ajmalicine and the antineoplastic agents vincristine and vinblastine. While biosynthesis of MIA precursors has been elucidated, conversion of the common MIA precursor strictosidine to MIAs of different families, for example ajmalicine, catharanthine or vindoline, remains uncharacterized. Deglycosylation of strictosidine by the key enzyme Strictosidine beta-glucosidase (SGD) leads to a pool of uncharacterized reaction products that are diverted into the different MIA families, but the downstream reactions are uncharacterized. Screening of 3600 EMS (ethyl methane sulfonate) mutagenized C. roseus plants to identify mutants with altered MIA profiles yielded one plant with high ajmalicine, and low catharanthine and vindoline content. RNA sequencing and comparative bioinformatics of mutant and wildtype plants showed up-regulation of SGD and the transcriptional repressor Zinc finger Catharanthus transcription factor (ZCT1) in the mutant line. The increased SGD activity in mutants seems to yield a larger pool of uncharacterized SGD reaction products that are channeled away from catharanthine and vindoline towards biosynthesis of ajmalicine when compared to the wildtype. Further bioinformatic analyses, and crossings between mutant and wildtype suggest a transcription factor upstream of SGD and ZCT1 to be mutated, leading to up-regulation of Sgd and Zct1. The crossing experiments further show that biosynthesis of the different MIA families is differentially regulated and highly complex. Three new transcription factors were identified by bioinformatics that seem to be involved in the regulation of Zct1 and Sgd expression, leading to the high ajmalicine phenotype. Increased cathenamine reductase activity in the mutant converts the pool of SGD reaction products into ajmalicine and its stereoisomer tetrahydroalstonine. The stereochemistry of ajmalicine and tetrahydroalstonine biosynthesis in vivo and in vitro was further characterized. In addition, a new clade of perakine reductase-like enzymes was identified that reduces the SGD reaction product vallesiachotamine in a stereo-specific manner, characterizing one of the many reactions immediately downstream of SGD that determine the different MIA families. This study establishes that RNA sequencing and comparative bioinformatics, in combination with molecular and biochemical characterization, are valuable tools to determine the genetic basis for mutations that trigger phenotypes, and this approach can also be used for identification of new enzymes and transcription factors.