Identification and characterization of a FAD-dependent putrescine N-hydroxylase (GorA) from Gordonia rubripertincta CWB2

Publication date: Available online 28 August 2016 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Catherine O. Esuola, Olubukola O. Babalola, Thomas Heine, Ringo Schwabe, Micheal Schlömann, Dirk Tischler 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 51.4kDa. It has been successfully cloned, overexpressed, and purified as a soluble flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) dependent His10-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±0.1μM and 0.266±0.011s−1, respectively, whereas the hydroxylation assay showed ...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research