STRAWBERRY DIOXYGENASE: STRUCTURAL AND FUNCTIONAL STUDY BY HOMOLOGY MODELING

Abstract

Strawberry, in technical terms, is an aggregate accessory fruit and is being used in food and beverages, besides this, strawberry pigment extracts can be used as natural acid/base indicator due to different color of the conjugate acid and bases in the pigment. Strawberries contain Fisetin, an antioxidant which can be studied in relation to Alzheimers’ disease and to kidney failure resulting from diabetes. Strawberries are of high nutritional value, and it is also a good source of flavonoids. Strawberry contains chemical compounds i.e. dimeric ellagitannin agrimoniin, which is an isomer of sanguiin. Anaphylactoid reactions to the consumption of strawberries have also been reported, which indicate that strawberry contains some allergen also. Strawberry has potential health benefits. Strawberry leaves and fruit extracts are useful in cardiovascular diseases and other disorders. Strawberry consumption decreases high blood pressure and reduces the risk of heart disease. Polyphenols in strawberries help regulate blood sugar response with decreased risk of type 2 diabetes. Strawberry is a good source of antioxidants which can help boost immune system and fight free radicals. The flavonol and other secondary metabolites such as anthocyanidins, quercetin, kempferol, succinate, fumarate etc. have various functions including anti-inflammatory, decrease the effects of Alzheimer’s disease, relieve rheumatoid arthritis and the acids might help remove stains from teeth. Strawberry extracts are very low in saturated fats, cholesterol and sodium. Strawberries help prevent macular degeneration, enhance memory functions and prevent types of cancers including esophageal, colon, breast, ovarian, cervical and lung cancer. These health benefits indicate that strawberries could be a potential dietary supplement. In strawberries, dioxygenases are reported to be essential for oxidative transformation reactions including fruit ripening, flavor biogenesis and plant defense responses against pathogens. A dioxygenases is an enzyme which incorporates both the atoms of molecular oxygen into kinds of substrates, undergoing various types of mechanisms. One of the most important functions of dioxygenases would be cleavage of aromatic rings, which is an important step in the degradation of aromatic compounds. Based on the substrate, dioxygenases could be further classified broadly into carotenoid cleavage dioxygenases (CCDs) and specifically into 9-cis-epoxycarotenoid cleavage dioxygenases (NCEDs), which catalyzes the rate limiting step in ABA biosynthesis; supposed oxidative cleavage of iii the carbon-carbon double bond of 9-cis-violaxanthin or 9-cis-neoxanthin. In maize (Zea maize), VP14 (Viviparous 14) is a kind of 9-cis-epoxycarotenoid dioxygenase and is one of the first characterized NCED through X-ray crystallography. NCEDs including VP14 are key regulators that determine ABA levels, which in turn control ABA-regulated processes. Homologs of VP14 have been identified in other plant species as well. Similar proteins have also been identified in variety of other plants, prokaryotes and animals. These results along with those studies into the physiological effects of ABA or apocarotenoid biosynthesis, laid the foundation for a more complete understanding of ABA biosynthesis and apocarotenoid biosynthesis or retinoid functions. Yet, although, various mechanisms for the rate limiting step in ABA biosynthesis and the 9-cis-carotenoid double bond cleavage have been proposed but the structure and the natural properties of specific determinants of specificity of dioxygenase pertaining to substrate specificity related amino acid motifs or encoding ring motifs are subject of intensive study. To characterize the cleavage reaction mechanism, structural and other studies of strawberry dioxygenase would be of value. If the structure of the protein and the coordination of the iron in the active site are determined and its consistency with previously determined dioxygenase structures, also further analysis of the substrate activity relationships and identification of potential inhibitor compounds could provide clues to classify the strawberry dioxygenase and methods to understand how CCDs and NCEDs work. The dioxygenase – substrate structure model can also be used for the development of therapeutics as this will promote strawberry as a useful dietary supplement. Keeping in view the above facts, the present study aims at understanding the strawberry dioxygenase structure by homology modeling approach, with the following objectives-  Strawberry dioxygenase structure model analyses  Strawberry dioxygenase and substrate activity interactions  Identification of strawberry dioxygenase inhibitors The Thesis is divided into 4 Chapters. Chapter 1 reviews the articles related to strawberry fruit ripening, flavor biogenesis; oxidative transformation and self hydroxylation reactions, herbicides and herbicide degradation and available dioxygenase structures, such as maize VP14, 4-HPPD, Tryptophan-2,3-dioxygenase, Indoleamine-2,3-dioxygenase and RPE65. iv Chapter 2 describes Strawberry dioxygenase sequence and structural analysis. Dioxygenase amino acid sequence was retrieved from the NCBI database and after various analysis i.e. transit peptide, transmembrane fragments, hydropathy, conserved domain and motifs, post translational modification sites, e.g. glycosylation, amidation and phosphorylation etc., the template based structure model was generated by using different servers. The Structure model was further analyzed for quality assessment. The membrane orientation and 2D representation of dioxygenase shows that the N- and C-terminals lie in the intracellular side and dioxygenase structure is tilted by an angle (46±3°) from the membrane surface. The secondary structure and topology analysis indicates that the dioxygenase structure contains an α-helical domain and a β-propeller structure that is also consistent with dioxygenase structures of this class. The metal coordination or Fe2+ ion centre is consistent with other dioxygenase structures as the Fe2+ is harbored on the central axis of the tunnel that runs across the complete structure by four octahedrally bound histidine residues (His225, His273, His339, His529) located on long loops. This type of Fe2+ coordination is found only in a few enzymes such as superoxide dismutase, photosystem II, fumerate reductase. All attempts to refine the structure shows that molecular oxygen is the most likely ligand at this position from a crystallographic as well as from a mechanistic point of view. The structure and coordination of strawberry dioxygenase were compared with Human tryptophan-2,3-dioxygenase and Indoleamine-2,3-dioxygenase which indicated that its human counterparts are mainly membrane proteins with several transmembrane segments while dioxygenase is a monotopic membrane protein. Also, consistently the substrates are likely to be trapped within the transmembrane domain completely or minimally occluded from the intracellular side yet solvent exposed from the extracellular side through a pore of diameter 8-40 A°, which is presumably not too wide for substrate to escape. The structure also indicates that α-helical domain may be interacting with the membrane surface, acting as a gate for substrate entry, which is consistent with Maize VP14 and it is assumed that other dioxygenase structures would contain the same conformation and have similar substrate interaction mechanisms. The study also identifies specific amino acid residues based on sequence analysis, which may be involved in self hydroxylation reactions and enantiomeric and stereo selectivity of substrates. Chapter 3 describes specific substrate binding domains in the dioxygenase structure. These substrate binding domains also provide reasons to assume that dioxygenase plays a very central role in the signaling pathway that connects catabolic processes to the plant defense v triggered by different allergens and also to the activated peripheral immune response during diseased conditions in humans. The study includes dioxygen interaction with dioxygenase which is the most fundamental interaction in these classes of enzymes. Also, some of the important interacting proteins and substrates include Allergen and lipids, Epoxycholesterol, Cell adhesion proteins, Ser/Thr kinases, Actin like ATPase, Dicer like protein, Peptide binding protein, Myosin ATPase and Immunoglobulin protein. These binding domains are imperative that it can be hypothesized that plant hormone interactions with dioxygenase in plants could pave the way to find potential new inhibitors or herbicides and the structure model could provide insights into designing targeted drugs pertaining to the treatment of various diseases and related conditions. Chapter 4 describes dioxygenase interactions with plant hormone Abscisic acid receptor (PYL9) and other selective herbicide inhibitors including Indoleamine imidazole, 2,4-D, Dicamba etc. Compounds containing amines, imidazole are potential dioxygenase inhibitors such as 2,4-D and Dicamba, which are synthetic versions of plant hormone Auxin. It is quite known for some time that these compounds act on methyl group of substrates. The correct positioning of aromatic ring of Hydroxamic acid or Abamine derivative compounds in the active site serves the basis of enzyme inhibition as the positioning of aromatic ring in the active site competes with the substrate and could be a common mechanism for competitive inhibition of these group of enzymes. Some possible modifications of specific amino acid residues such as tyrosine hydroxylation and phosphorylation, glutamine and asparagine deamination are involved in the His1-Glu/Asp-His2 mechanism, which is widely applicable and it provides reasons to assume that self hydroxylation reactions are not very uncommon uncoupled reaction in this class of enzymes. Chapter 5 describes overall methods used for analyzing transmembrane segments, transit peptide, folds and motifs in strawberry dioxygenase protein sequence retrieved from database. The structural model was generated and analyzed by using different servers and tools. Finally, the main conclusions of the present study are summarized in Chapter 6. The structure of strawberry dioxygenase is consistent with other characterized structures of this family of dioxygenases. The structure of strawberry dioxygenase suggests some substrate and protein binding domains based on which potential protein partners and inhibitor vi compounds are identified. The study also suggests the health benefits of strawberry dioxygenase by dioxygenase and possible drug substrate interactions. The structural similarity of strawberry dioxygenase and its human counterparts Tryptophan-2,3-dioxygenase and Indolamine-2,3-dioxygenase and Integrin receptors and substrate binding domains within the strawberry dioxygenase structure indicate that some dioxygenase functions may be common in plant and animal or human enzymes. This finding provide a reason to speculate the role played by dioxygenases in peripheral immune responses and it suggests that dioxygenase could be acting as an important node in the signaling pathway triggered by receptor tyrosine kinase or Integrin receptors, which sense mechanical constraints within the cell. It is hypothesized that some specific amino acid residues including arginine and tryptophan could act as dietary amino acids and may be involved in dioxygenase mediated regulation of Tregs and dendritic cells. This study on the basis of structure model also suggests a predictive role of dioxygenases under Hypoxia or similar conditions, Angiogenesis, Cell differentiation, Cell proliferation and migration; Tissue morphogenesis, Tumor growth i.e. Solid and circulating tumors and underlying pathways and Poly(ADP-ribose)polymerase mediated DNA damage associated cell cycle control. The study also predicts the role of dioxygenases in neuronal development and in the Axon guidance pathway. Moreover, based on the structural homology and dioxygenase structure several targeted drugs can be selected for analyses and in terms of their modes of action the underlying pathways can be studied in detail. Such targeted therapeutics would benefit patients with different type of diseases, including metabolic symptomatic disorders, neurogenerative diseases and certain forms of cancers.