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Molecular cloning, gene expression analysis, and in silico characterization of UDP‐N‐acetylglucosamine pyrophosphorylase from Bombyx mori
Author(s) -
Palaka Bhagath Kumar,
Velmurugan Ilavarasi Anbumani,
Sapam Tuleshwori Devi,
Kotapati Kasi Viswanath,
Nallala Venkata Satyanarayana,
Khan Mohd. Babu,
Ampasala Dinakara Rao
Publication year - 2019
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1802
Subject(s) - bombyx mori , biology , in silico , biochemistry , complementary dna , protein data bank (rcsb pdb) , microbiology and biotechnology , peptide sequence , molecular cloning , sequence alignment , sequence analysis , gene
The present study was aimed to explore the molecular and structural features of UDP‐N‐acetylglucosamine pyrophosphorylase of Bombyx mori ( Bm UAP), an essential enzyme for chitin synthesis in insects. The Bm UAP cDNA sequence was cloned and expression profiles were monitored during the molting and feeding stages of silkworm larvae. The effect of 20‐hydroxyecdysone (20E) on Bm UAP expression, and on silkworm molting was studied, which revealed that 20E regulates its expression. Multiple sequence alignment of various pyrophosphorylases revealed that the residues N223, G290, N327, and K407 of human UAP (PDB ID: 1JV1) were found to be highly conserved in Bm UAP and all other eukaryotic UAPs considered for the study. Phylogenetic analysis inferred that the UAPs possess discrete variations in primary structure among different insect Orders while sharing good identity between species of the Order. The structure of Bm UAP was predicted and its interactions with uridine triphosphate, N‐acetylglucosamine‐1‐phosphate, and UDP‐N‐acetylglucosamine were analyzed. Virtual screening with a library of natural compounds resulted in five potential hits with good binding affinities. On further analysis, these five hits were found to be mimicking substrate and product, in inducing conformational changes in the active site. This work provides crucial information on molecular interactions and structural dynamics of insect UAPs.