نوع مقاله : مقاله پژوهشی
نویسندگان
گروه علوم باغبانی و مهندسی فضای سبز، دانشکده کشاورزی، دانشگاه تهران، کرج، ایران
چکیده
کلیدواژهها
موضوعات
عنوان مقاله [English]
نویسندگان [English]
Crocus sativus L. is considered as one of the richest sources of apocarotenoids, including crocin, picrocrocin, and safranal. The oxidative breakdown of carotenoids generates apocarotenoids. Glycosylation is the final step of crocin biosynthesis which is crucial due to its role in pigment solubility in water and changes the chemical properties and bioactivity of the molecule. In this study, the protein sequence of UGTs in saffron and their homologs in other plants were analyzed from different points of view, including phylogeny analysis and motif identification, functional divergence analysis, and structural analysis. The present study focused on UGTs responsible for primary and secondary glycosylation in crocin production and picrocrocin glycosylation in plants where crocin is found. Also, the evolutionary relationship of the UGT protein family in Crocus and other plants was investigated, including type I and type II functional divergence. Phylogeny analysis showed that UGTs that carry out primary glycosylation and UGTs that carry out secondary glycosylation were placed in two groups with the highest functional distance. Motifs were group-specific and amino acids with a high functional divergence coefficient were identified in those motifs, which can be attributed to the functional difference of these sequences. These findings may facilitate future researches aimed at characterizing the function of these genes.
کلیدواژهها [English]
Extended Abstract
Introduction
Glycosyltransferases catalyze the transfer of a sugar residue from nucleotide-sugar donors to various acceptor molecules. These enzymes change the hydrophilicity, chemical properties, and bioactivity of the molecules. The process of glycosylation is critical in the food and pharmaceutical industries. Prediction of functional residues in a protein is crucial because these residues can be attributed to new functions, change the protein properties, define protein families and subfamilies, or identify the occurrence of an innovation. A phylogenetic analysis of a protein family and functional divergence analysis is valuable for identifying conserved and divergent regions that may offer insights into potential functions.
Material and Methods
In the current study, we placed particular emphasis on UGTs (UDP-glucuronosyltransferases), which play a critical role in both primary and secondary glycosylation during crocin biosynthesis. Two distinct datasets were gathered. The first dataset contained UGT protein sequences specifically identified within the Crocus genus. The second dataset comprised homologous UGT protein sequences retrieved from NCBI using the C. sativus UGTs gene sequence as a query in other plant species. On the first data set, phylogeny and motif analysis and on the second data set, phylogeny, motif, functional divergence, and structural similarity analysis were performed. The phylogeny tree was constructed using MrBayes 3.1.2 software. DIVERGE 3.0 software was utilized to identify functional divergence among members of the UGT protein family. Some UGTs found in plants containing crocin were compared for structural analysis. The SWISS-MODEL server was used for structure prediction, the PyMOL software was employed for structure visualization, and the Dali server was utilized to compare the degree of structural similarity among the desired UGT structures.
Result and discussion
The study focused on UGT enzymes in the Crocus genus, specifically comparing those responsible for primary and secondary glycosylation in crocin production. According to the phylogeny analysis, UGT proteins were classified into different subfamilies, forming separate branches on the tree. Results revealed four distinct groups within Crocus and six when including sequences from other plants in the phylogenetic tree analysis. In both phylogenetic trees, the primary and secondary glycosylation groups were distinctly separated from the beginning, indicating the difference between these two groups.
Using MEME software, 15 shared motifs were identified among these sequences, which likely play crucial roles in protein specificity and function. Based on the position and presence of motifs in sequences, it has been concluded that sequences grouped together likely share similar functions. On the other hand, sequences grouped in different clusters perform distinct functions, which can be justified by the presence of different motifs within them. When UGTs of Crocus were studied alongside other plants, motifs 10 and 11 were found in the primary glycosylation group, overlapping with motifs 5 and 7 in the primary glycosylation group identified in the first analysis. This overlap may indicate the significance of these motifs and their potential role in the functional specificity of this group.
Based on previous researches, while the C-terminal region of UDP-glucuronosyltransferases (UGTs) often interacts with the sugar donor group, the N-terminal region interacts in substrate recognition of the sugar acceptor group. Additionally, crystal structures of UGTs have shown that the N-terminal region is less protected compared to the C-terminal region, which correlates with the diversity of UGT receptors. In the current study, specific motifs at positions 7 (first analysis) or 11 (second analysis) in primary glycosylation groups, and motif 13 (second analysis) in secondary glycosylation groups located at the N-terminal region. According to previous studies, these motifs may contribute to substrate specificity between primary and secondary glycosylation groups.
In the present study, the functional divergence coefficient of type 1 was significantly greater than zero, indicating a substantial divergence pattern among the subfamilies examined. However, the functional divergence coefficient of type 2 was very low. These results suggest a predominant pattern of functional divergence type 1 for distinguishing between the subfamilies under investigation, and selective pressure specific to certain sites is likely to play a significant role in most UGT genes, leading to the evolution of specific subfamily functions following divergence.
Conclusion
Phylogeny analysis showed that UGTs that carry out primary glycosylation and UGTs that carry out secondary glycosylation were placed in two separate groups. These two groups had high functional distance. In each group, motifs were found specific to the same group. In these specific motifs, amino acids with a high functional divergence coefficient were identified, which can be attributed to the functional difference of these sequences.
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