Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95

Rao, Saroja Narsing; Mohan, Anil H. Shyam; Srividya, D.; Supreetha, K.

doi:10.1016/j.pep.2019.01.006

Cited by 3 publications

(8 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These NMO need either NAD or NADPH for catalysis [ 5 ]. In our previous study, chaperone-mediated expression of Sp PMO was carried out along with kinetic studies using putrescine and lysine substrates [ 8 ]. Even so, Sp PMO is not explored exhaustively.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, the instability index of putrescine monooxygenase group has been observed to be between 37 and 48. The unstable nature of these proteins can be noted during heterologous expression studies of these proteins [ 4 , 8 , 29 ], wherein they have to be tagged with a soluble protein like MBP or co-expressed with a chaperone for soluble expression. Although the instability index of lysine monooxygenases falls within 40, the researchers have faced difficulties in soluble expression of these enzymes in E. coli [ 5 ].…”

Section: Resultsmentioning

confidence: 99%

“…The amino acid sequence of putrescine monooxygenase from Shewanella putrefaciens-95 was retrieved from the previous work [ 8 ]. The other NMO sequences from Shewanella baltica , Shewanella putrefaciens 200, Shewanella putrefaciens CN32, Gordonia ruberipertincta , Streptomyces , Bordetella pertussis , Erwinia amylovora , Escherichia coli , Mycobacterium tuberculosis , Nocardia facinica , Pseudomonas aeruginosa , Kutzneria spp., Aspergillus fumigates , Amycolatopsis alba along with the representative sequences of Baeyer-Villiger monooxygenases (BVMO) from Xanthobacter flavus and flavin containing monooxygenases (FMO) from Homo sapiens and Methylophaga aminisulfidivorans were retrieved from the National Centre for Biotechnology Information (NCBI) website [ 11 ] (Supplementary data).…”

Section: Methodsmentioning

confidence: 99%

“…The quantity of N-hydroxyl putrescine formation in the presence and absence of inhibitors was assayed by a modified Csaky iodine oxidation protocol as done in our previous study [ 8 ]. The enzyme Sp PMO (1 μM) in 100 mM sodium phosphate buffer was incubated with putrescine (0–25 mM) and the inhibitors curcumin or niazirin (0–0.01 mM) for 10 min at 25°C and 1000 rpm shaking.…”

Section: Methodsmentioning

confidence: 99%

“…[ 6 , 7 ]. In our previous study, we have also co-expressed NMO from Shewanella putrefaciens (putresine monooxygenase— Sp PMO) along with GroES/EL chaperone in E. coli BL21 star (DE3) [ 8 ]. Shewanella putrefaciens is gram-negative bacteria usually present in shallow water bodies.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Mohan

Rao

Srividya

et al. 2022

Journal of Genetic Engineering and Biotechnology

Self Cite

View full text Add to dashboard Cite

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 (SpPMO) 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 SpPMO 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 SpPMO 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 SpPMO 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 SpPMO 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

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

Mohan

Rao

Srividya

et al. 2022

Journal of Genetic Engineering and Biotechnology

Self Cite

View full text Add to dashboard Cite

show abstract

Flavin-dependent N-hydroxylating enzymes: distribution and application

Mügge

Heine

Baraibar

et al. 2020

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

Amino groups derived from naturally abundant amino acids or (di)amines can be used as "shuttles" in nature for oxygen transfer to provide intermediates or products comprising NO functional groups such as N-hydroxy, oxazine, isoxazolidine, nitro, nitrone, oxime, C-, S-, or N-nitroso, and azoxy units. To this end, molecular oxygen is activated by flavin, heme, or metal cofactorcontaining enzymes and transferred to initially obtain N-hydroxy compounds, which can be further functionalized. In this review, we focus on flavin-dependent N-hydroxylating enzymes, which play a major role in the production of secondary metabolites, such as siderophores or antimicrobial agents. Flavoprotein monooxygenases of higher organisms (among others, in humans) can interact with nitrogen-bearing secondary metabolites or are relevant with respect to detoxification metabolism and are thus of importance to understand potential medical applications. Many enzymes that catalyze N-hydroxylation reactions have specific substrate scopes and others are rather relaxed. The subsequent conversion towards various NO or N-N comprising molecules is also described. Overall, flavin-dependent N-hydroxylating enzymes can accept amines, diamines, amino acids, amino sugars, and amino aromatic compounds and thus provide access to versatile families of compounds containing the NO motif. Natural roles as well as synthetic applications are highlighted. Key points • NO and N-N comprising natural and (semi)synthetic products are highlighted. • Flavin-based NMOs with respect to mechanism, structure, and phylogeny are reviewed. • Applications in natural product formation and synthetic approaches are provided.

show abstract

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase

et al. 2023

View full text Add to dashboard Cite

Chaperone-assisted expression and purification of putrescine monooxygenase from Shewanella putrefaciens-95

Cited by 3 publications

References 31 publications

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

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

Flavin-dependent N-hydroxylating enzymes: distribution and application

Validation of Lon Gene Disruption using Linear DNA Cassette by Crelox Mechanism in E. coli Strains: To Achieve Better Solubility of Putrescine Monooxygenase

Contact Info

Product

Resources

About