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

•GorA is the first biochemically characterized diamine N-hydroxylase.•GorA is a NADPH- and FAD-dependent soluble protein, but accepts to some extent other coenzymes.•GorA prefers diamines: putrescine > cadaverine > hexamethylenediamine.•Uncoupling of GorA in the presence of other substrates has been observed.•GorA seems to be involved in siderophore biosynthesis of Gordonia rubripertincta CWB2.

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 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 Km and kcat of 361.6 ± 0.1 μM and 0.266 ± 0.011 s−1, respectively, whereas the hydroxylation assay showed GorA with an apparent Km and kcat 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.

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