Expression in Escherichia coli of the flavin and the haem domains of Hansenula anomala flavocytochrome b2 (flavodehydrogenase and b2 core) and characterization of the recombinant proteins.
The flavin and haem domains of Hansenula anomala flavocytochrome b2 have been independently expressed in Escherichia coli. The flavin domain activity, studied only in the total cellular extract, owing to its instability, has characteristics very similar to those of the flavin domain obtained by proteolysis. The haem domain (r-core) has been purified to homogeneity and characterized in detail from spectroscopic and functional points of view. Spectral differences with respect to the domain produced by proteolysis (p-core) were found using resonance Raman and c.d. spectroscopy and have been interpreted in terms of changes in haem-protein interactions. However, this structural difference is functionally silent, since the r-core is able to reduce cytochrome c with the same efficiency as the proteolytic domain.
Silvestrini
M C
MC
Dipartimento di Scienze Biochimiche, Università di Roma La Sapienza, Italy.
Tegoni
M
M
Célerier
J
J
Desbois
A
A
Gervais
M
M
D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli; D005415:Flavins / Q000235:genetics*; D006418:Heme / Q000235:genetics*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013059:Spectrum Analysis, Raman
D005415:Flavins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome)