Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

Erik W. van Hellemond, Marianne van Dijk, Dominic P. H. M. Heuts, Dick B. Janssen, Marco W. Fraaije*

*Bijbehorende auteur voor dit werk

OnderzoeksoutputAcademicpeer review

34 Citaten (Scopus)
326 Downloads (Pure)


A gene encoding a putrescine oxidase (PuORh, EC was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K-M=8.2 mu M, k(cat)=26 s(-1)). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t (1/2) at 50 degrees C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuORh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuORh.

Originele taal-2English
Pagina's (van-tot)455-463
Aantal pagina's9
TijdschriftApplied Microbiology and Biotechnology
Nummer van het tijdschrift3
StatusPublished - mrt-2008

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