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Expression and Characterization of a Functional Leishmania Variant of Cytochrome b5 Reductase
Author(s) -
Barber Michael J,
Roma Glenn William,
Seyfang Andreas
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.634.3
Subject(s) - cytochrome b5 , nad+ kinase , enzyme kinetics , biochemistry , flavoprotein , reductase , enzyme , histidine , chemistry , flavin group , biology , microbiology and biotechnology , active site
Cytochrome b5 reductase (cb5r), a member of the FNR family of flavoprotein transhydrogenases, catalyzes the NADH‐dependent reduction of cytochrome b5 (cb5). The putative nucleotide sequence for the L. donovani enzyme was used to clone and generate a histidine‐tagged form of the soluble, catalytic domain of cb5r. The recombinant protein was purified to homogeneity and exhibited absorption and CD spectra comparable to the wild type rat cb5r diaphorase domain, suggesting similar protein folding and flavin incorporation. Initial‐rate kinetic studies showed a decreased turnover rate in the Leishmania diaphorase domain, (kcat of 283s‐1) for the NADH:FR as compared to the rat domain (800s‐1). Km for NADH was comparable to that of wild type rat cb5r. Thermal stability studies revealed decreased thermal stability (49oC) compared to that of the rat protein (55oC). Spectral binding studies indicated comparable affinity for and binding of H4NAD and NAD+ for both variants. Oxidation‐reduction potentials in both the absence (−275mV) and presence (−195mV) of NAD+ were comparable to values previously obtained for the corresponding rat domain (−274 mV and −190 mV). These results provide direct comparison of an enzyme from two divergent phylogenetic sources using identical expression systems and indicate a species‐based difference in the structural, spectroscopic, kinetic and thermodynamic properties of the enzyme.