NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability.
NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Songklod
S
Sarapusit
Chuanwu
C
Xia
Ila
I
Misra
Pornpimol
P
Rongnoparut
Jung-Ja P
JJ
Kim
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000852:Anopheles / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000235:genetics; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D017931:DNA Primers; D005486:Flavin Mononucleotide; D009251:NADPH-Ferrihemoprotein Reductase; D007930:Leucine