Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans.
FerB is a flavoenzyme capable of reducing quinones, ferric complexes and chromate. Its expression in Escherichia coli as a hexahistidine fusion resulted in a functional product only when the tag was placed on the C-terminus. The molecular mass values estimated by gel permeation chromatography were compatible with the existence of either dimer or trimer, whereas the light scattering data, together with cross-linking experiments that yielded exclusively monomer and dimer bands on dodecyl sulfate-polyacrylamide gels, strongly supported a dimeric nature of both native and tagged form of FerB. These two proteins also exhibited almost identical secondary structure as judged by Fourier transform infra red spectrometry. The presence of tag, however, shifted the temperature of thermal inactivation as well as the thermal denaturation curve towards lower temperatures. Despite somewhat lower thermal stability, the fusion protein is considered a better candidate for crystallization than the wild-type one due to a more negative value of its second optical viral coefficient.
Tesarík
Radek
R
Department of Vaccinology and Immunotherapy, Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, Czech Republic.
Sedlácek
Vojtech
V
Plocková
Jana
J
Wimmerová
Michaela
M
Turánek
Jaroslav
J
Kucera
Igor
I
D002152:Calorimetry, Differential Scanning; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005583:Fourier Analysis; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D009249:NADP; D009247:NADH, NADPH Oxidoreductases