In silico structural, phylogenetic and drug target analysis of putrescine monooxygenase from Shewanella putrefaciens-95

• Published in: Journal of Genetic Engineering and Biotechnology Vol. 20; no. 1; pp. 57 - 14
• Main Authors: Shyam Mohan, Anil H., Rao, Saroja Narsing, D., Srividya, Rajeswari, N.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 12.04.2022; Springer; Springer Nature B.V; Elsevier
• Subjects: Affinity; Amino acids; Analysis; Antibiotics; Bacteria; Binding; Biomedical Engineering and Bioengineering; Conserved sequence; Curcumin; E coli; Engineering; Enzymes; Escherichia coli; Flavin-adenine dinucleotide; Immunity; Inhibitors; Iodine; Maximum likelihood method; Metal oxide semiconductors; Microorganisms; Molecular docking; Monooxygenase; N-Hydroxylating monooxygenases; Naturally occurring inhibitors; Nucleotide sequence; Nutrients; Phylogenetic relationship; Phylogenetics; Phylogeny; Physicochemical characteristics; Physicochemical properties; Polyamines; Protein structure; Proteins; Putrescine; Secondary structure; Shewanella putrefaciens; Software; Substrate preferences; Substrate specificity; Substrates; Therapeutic targets; Three dimensional models; Trace elements; Tuberculosis; Whooping cough; India; Phylogenetic relationship; Naturally occurring inhibitors; Physicochemical characteristics; N-Hydroxylating monooxygenases; Molecular docking; Shewanella putrefaciens
• ISSN: 1687-157X; 2090-5920
• DOI: 10.1186/s43141-022-00338-z

Background The enormous and irresponsible use of antibiotics has led to the emergence of resistant strains of bacteria globally. A new approach to combat this crisis has been nutritional immunity limiting the availability of nutrients to pathogens. Targeting the siderophore biosynthetic pathway that helps in iron acquisition, an essential microelement in the bacterial system has been the topic of interest in recent days that backs the concept of nutritional immunity. Supporting this view, we have chosen to study a key enzyme in the biosynthetic pathway of putrebactin called putrescine monooxygenase ( Sp PMO) from Shewanella putrefaciens. In our previous study, we co-expressed putrescine monooxygenase recombinantly in Escherichia coli BL21 Star (DE3). The bioinformatic analysis and screening of inhibitors will broaden the scope of Sp PMO as a drug target. Results In the present study, we have analysed the physicochemical properties of the target enzyme and other N-hydroxylating monooxygenases (NMOs) using ExPASy server. The target enzyme Sp PMO and most of the selected NMOs have a slightly acidic isoelectric point and are medially thermostable and generally insoluble. The multiple sequence alignment identified the GXGXX(N/A), DXXXFATGYXXXXP motives and conserved amino acids involved in FAD binding, NADP binding, secondary structure formation and substrate binding. The phylogenetic analysis indicated the distribution of the monooxygenases into different clades according to their substrate specificity. Further, a 3D model of Sp PMO was predicted using I-TASSER online tool with DfoA from Erwinia amylovora as a template. The model was validated using the SAVES server and deposited to the Protein Model Database with the accession number PM0082222. The molecular docking analysis with different substrates revealed the presence of a putrescine binding pocket made of conserved amino acids and another binding pocket present on the surface of the protein wherein all other ligands interact with high binding affinity. The molecular docking of naturally occurring inhibitor molecules with Sp PMO 3D model identified curcumin and niazirin with 1.83 and 2.81 μM inhibition constants as two promising inhibitors. Further studies on kinetic parameters of curcumin and niazirin inhibitors in vitro determined the Ki to be 2.6±0.0036 μM and 18.38±0.008 μM respectively. Conclusion This analysis will help us understand the structural, phylogenetic and drug target aspects of putrescine monooxygenase from Shewanella putrefaciens- 95 in detail. It sheds light on the precautionary measures that can be developed to inhibit the enzyme and thereby the secondary infections caused by them. Graphical abstract Highlights The physicochemical properties of putrescine monooxygenase from Shewanella putrefaciens- 95 were compared with other known N-hydroxylating monooxygenases. The phylogenetic analysis of Sp PMO and other selected class B monooxygenases clearly classified NMOs according to their substrate affinity. The molecular docking analysis of various substrates to Sp PMO enzyme showed the presence of two binding pockets. Curcumin and niazirin were identified as two potential natural inhibitors to Sp PMO enzyme with 2.6±0.0036 μM and 18.38±0.008 μM inhibition constants respectively.