Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540.
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.
van Hellemond
Erik W
EW
Laboratory of Biochemistry, University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
van Dijk
Marianne
M
Heuts
Dominic P H M
DP
Janssen
Dick B
DB
Fraaije
Marco W
MW
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; C022488:putrescine oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors