Towards a new interaction enzyme:coenzyme.
Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.
Martínez-Júlvez
Marta
M
Departamento de Bioquímica y Biología Molecular y Celular and Institute of Biocomputation and Physics of Complex Systems (BiFi), Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Tejero
Jesús
J
Peregrina
José R
JR
Nogués
Isabel
I
Frago
Susana
S
Gómez-Moreno
Carlos
C
Medina
Milagros
M
D017033:Anabaena / Q000201:enzymology*; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013057:Spectrum Analysis; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D009249:NADP; D005287:Ferredoxin-NADP Reductase