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

Applied Microbiology and Biotechnology
Erik W van HellemondMarco W Fraaije

Abstract

A gene encoding a putrescine oxidase (PuORh, EC 1.4.3.10) 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 (KM=8.2 microM, kcat=26 s(-1)). PuORh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuORh is a reasonably thermostable enzyme with t1/2 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.

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Citations

May 10, 2011·Applied Microbiology and Biotechnology·Jens Schneider, Volker F Wendisch
Aug 3, 2013·Systematic and Applied Microbiology·Alexander FosterMark A Keane
Apr 14, 2011·Biochemistry·Malgorzata M KopaczAndrea Mattevi
Sep 5, 2013·Journal of Agricultural and Food Chemistry·Jae-Ick LeeYoung-Wan Kim
Aug 6, 2008·The Journal of Biological Chemistry·Erik W van HellemondMarco W Fraaije
Jun 2, 2009·Journal of Bacteriology·Dirk TischlerMichael Schlömann
May 4, 2013·Applied Microbiology and Biotechnology·Willem P DijkmanMarco W Fraaije
Apr 2, 2013·Enzyme and Microbial Technology·Alexander FosterMark A Keane
Mar 31, 2015·Journal of Agricultural and Food Chemistry·Sara CallejónIsabel Pardo
May 5, 2011·Journal of Biotechnology·Luaine BandounasHarald J Ruijssenaars
Jul 26, 2014·The FEBS Journal·Malgorzata M KopaczMarco W Fraaije
Jun 22, 2015·Applied Biochemistry and Biotechnology·Zhenming ChenLinshu Jiang
Oct 22, 2020·Scientific Reports·Daniele Salvi, Paraskevi Tavladoraki
Jun 9, 2021·Langmuir : the ACS Journal of Surfaces and Colloids·Nilan J B KamathewattaCindy L Berrie
Jul 16, 2021·Frontiers in Bioengineering and Biotechnology·Luciana Fernandes BritoTrygve Brautaset
Sep 15, 2020·Langmuir : the ACS Journal of Surfaces and Colloids·Nilan J B KamathewattaCandan Tamerler
Apr 27, 2010·Archives of Biochemistry and Biophysics·Michelle Henderson Pozzi, Paul F Fitzpatrick

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Datasets Mentioned

BETA
EU240877

Methods Mentioned

BETA
electrophoresis
PCR
deamination

Software Mentioned

CHPmodels
Origin
ORF
BLAST

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