ABSTRACT
Allosteric feedback inhibition of the committed step in amino acid biosynthetic pathways is a major concern for production of amino acids at industrial scale. Anthranilate synthase (AS) catalyzes the first reaction of tryptophan biosynthetic pathway found in microorganisms and is feedback inhibited by its own product i.e. tryptophan. Here, we identified new mutant sites in AS using computational mutagenesis approach. MD simulations (20 ns) followed by MMPBSA and per residue decomposition energy analysis identified seven amino acid residues with best binding affinity for tryptophan. All 19 mutant structures were generated for each identified amino acid residue followed by simulation to evaluate effect of mutation on protein stability. Later, molecular docking studies were employed to generate mutant-tryptophan complex and structures with binding energies (kcal/mol) much higher than wild-type AS were selected. Finally, two mutants i.e., S37W and S37H were identified on the basis of positive binding scores and loss of tryptophan binding inside pocket. Further, MD simulations run for 200 ns were performed over these mutant-tryptophan complexes followed by RMSD, RMSF, radius of gyration , solvent accessible surface area , intra-protein hydrogen bond numbers, principal component analysis, free energy landscape (FEL) and secondary structure analysis to rationale effect of mutations on stability of protein. Cross correlation analysis of mutant site amino acids (S37W) with key residues of catalytic site (G325, T326, H395 and G482) was done to evaluate the effect of mutations on catalytic site conformation. Current computational mutagenesis approach predicted two mutants S37W and S37H with proposed deregulated feedback inhibition by tryptophan and retained catalytic activity.Communicated by Ramaswamy H. Sarma.
ABSTRACT
Background: Species of the genus Monascus are economically important and widely used in the production of food colorants and monacolin K. However, they have also been known to produce the mycotoxin citrinin. Currently, taxonomic knowledge of this species at the genome level is insufficient. Methods: This study presents genomic similarity analyses through the analysis of the average nucleic acid identity of the genomic sequence and the whole genome alignment. Subsequently, the study constructed a pangenome of Monascus by reannotating all the genomes and identifying a total of 9,539 orthologous gene families. Two phylogenetic trees were constructed based on 4,589 single copy orthologous protein sequences and all the 5,565 orthologous proteins, respectively. In addition, carbohydrate active enzymes, secretome, allergic proteins, as well as secondary metabolite gene clusters were compared among the included 15 Monascus strains. Results: The results clearly revealed a high homology between M. pilosus and M. ruber, and their distant relationship with M. purpureus. Accordingly, all the included 15 Monascus strains should be classified into two distinctly evolutionary clades, namely the M. purpureus clade and the M. pilosus-M. ruber clade. Moreover, gene ontology enrichment showed that the M. pilosus-M. ruber clade had more orthologous genes involved with environmental adaptation than the M. purpureus clade. Compared to Aspergillus oryzae, all the Monascus species had a substantial gene loss of carbohydrate active enzymes. Potential allergenic and fungal virulence factor proteins were also found in the secretome of Monascus. Furthermore, this study identified the pigment synthesis gene clusters present in all included genomes, but with multiple nonessential genes inserted in the gene cluster of M. pilosus and M. ruber compared to M. purpureus. The citrinin gene cluster was found to be intact and highly conserved only among M. purpureus genomes. The monacolin K gene cluster was found only in the genomes of M. pilosus and M. ruber, but the sequence was more conserved in M. ruber. Conclusion: This study provides a paradigm for phylogenetic analysis of the genus Monascus, and it is believed that this report will lead to a better understanding of these food microorganisms in terms of classification, metabolic differentiation, and safety.
ABSTRACT
BACKGROUND: Long intergenic non-protein coding RNA 707 (LINC00707) has been identified as a cancer-associated long non-coding RNA (lncRNA) in a variety of cancers. However, the functions and molecular mechanisms of LINC00707 in esophageal squamous cell carcinoma (ESCC) are still unclear. METHODS: The expression of LINC00707 in esophageal cancer (ESCA) and ESCC tissues was determined by online tools, RNA-sequence (RNA-seq) dataset, and quantitative real time polymerase chain reaction (qRT-PCR). The associations between LINC00707 expression and clinicopathologic features and prognosis were investigated. Furthermore, the expression of LINC00707 in ESCC cell lines was determined by qRT-PCR. Then, using LncACTdb 2.0 database, combined with loss-of-function assay verification, we investigated the biologic role of LINC00707 in ESCC cell growth, apoptosis, invasion, and migration by CCK-8, colony formation, flow cytometry and transwell assays. Finally, western blot was used to evaluate the regulatory effect of LINC00707 on PI3K/Akt signaling pathway. RESULTS: Increased LINC00707 expression was exhibited in ESCC tissues and cell lines. High expression of LINC00707 was positively associated with higher tumor-node-metastasis (TNM) stage and lymph node metastasis. Furthermore, LINC00707 expression was significantly higher in patients who drink alcohol, have lymph node metastasis, and harbor higher tumor stage. In addition, Kaplan-Meier survival analysis and receiver operating characteristic (ROC) curve confirmed the feasibility of LINC00707 as a prognostic signature or diagnostic marker. Functional experiments showed that LINC00707 downregulation suppressed ESCC cell proliferation, and metastasis, and induced ESCC cell apoptosis. Mechanistic investigation demonstrated that LINC00707 activated the PI3K/Akt signaling pathway in ESCC cells. CONCLUSIONS: Our findings suggest LINC00707 functions as an oncogenic lncRNA in ESCC, and imply that LINC00707 may be a promising prognostic biomarker and therapeutic target for ESCC patients.
