Identification and characterization of a FAD-dependent putrescine N-hydroxylase (GorA) from Gordonia rubripertincta CWB2
| Autor: | Olubukola Oluranti Babalola, Thomas Heine, Micheal Schlömann, Ringo Schwabe, Dirk Tischler, Catherine Oluwakemi Esuola |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Flavin adenine dinucleotide chemistry.chemical_classification Stereochemistry Process Chemistry and Technology 030106 microbiology Bioengineering NADPH oxidation Monooxygenase Biochemistry Catalysis Hydroxylation 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Enzyme chemistry Putrescine NAD+ kinase Nicotinamide adenine dinucleotide phosphate |
| Zdroj: | Journal of Molecular Catalysis B: Enzymatic. 134:378-389 |
| ISSN: | 1381-1177 |
| Popis: | A putrescine N -hydroxylase from Gordonia rubripertincta CWB2 (GorA), a microbial N -hydroxylating monooxygenase (NMO), specific for a range of diamines (putrescine > cadaverine > hexamethylenediamine) was identified. This NMO clustered together with some known but yet to be characterized diamine NMOs which are RhbE, from Sinorhizobium meliloti 1021; AlcA, from Bordetella bronchiseptica RB50, and DesB, from Streptomyces scabiei 87-22. It comprises 459 amino acids in length and has approximately a molecular weight of 51.4 kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His 10 -tagged protein using Escherichia coli as the cloning and expression host and pET16bP as vector. The NAD(P)H oxidation assay and a hydroxylation assay were used to assess its biochemical properties. The pH optimum is between the range of 7.0–8.0 in a potassium phosphate buffer. 1,4-diaminobutane (putrescine) was the best substrate concerning GorA activity. With the NADPH oxidation assay, the kinetic parameters of this enzyme showed an apparent K m and k cat of 361.6 ± 0.1 μM and 0.266 ± 0.011 s −1 , respectively, whereas the hydroxylation assay showed GorA with an apparent K m and k cat of 737.1 ± 0.1 μM and 0.210 ± 0.001 s −1 . These activity data were obtained of kinetic experiments from fixing FAD and NADPH and varying the concentration of 1,4-diaminobutane. Thus this is the first diamine N -hydroxylating monooxygenase characterized with a physiological role in siderophore biosynthesis. |
| Databáze: | OpenAIRE |
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