Overexpression in E. coli of the complete petH gene product from Anabaena: purification and properties of a 49 kDa ferredoxin-NADP+ reductase.
The complete petH gene product from Anabaena PCC 7119 has been overexpressed in E. coli and purified in order to determine the influence of the N-terminal extension on the interaction of ferredoxin-NADP+ reductase with its substrates. The intact 49 kDa FNR can be easily purified in a two-step procedure using batch extraction with DEAE-cellulose followed by Cibacron blue-Sepharose chromatography of the proteins unbound to DEAE. Isoelectric focusing of FNR shows several forms, with the major band at pH 6.26. The presence of the N-terminal extension increases the K(m) of FNR for NADPH by 4-fold and by 16.4-fold in the reduction reactions of DCPIP and cytochrome c. However, the K(m) for ferredoxin is 12-fold lower in the reaction catalyzed by the 49 kDa FNR than with the 36 kDa protein. This indicates that the presence of the third domain favours the interaction of FNR with ferredoxin, possibly due to the more positive net charge of the N-terminal extension. Comparable rate constants for both enzymes, were obtained for the photoreduction of NADP+ using photosynthetic membranes and also using rapid kinetic techniques. Slightly different ionic strength dependences of the rate constants were obtained, nevertheless, for both forms of the enzyme. These are a consequence of the structural differences that the proteins show at the N-terminal and of their effect on the interaction with ferredoxin.
Martínez-Júlvez
M
M
Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Spain.
Hurley
J K
JK
Tollin
G
G
Gómez-Moreno
C
C
Fillat
M F
MF
D017033:Anabaena / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D005420:Flavoproteins; D015870:Gene Expression / Q000235:genetics; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013053:Spectrophotometry
D001426:Bacterial Proteins; D017931:DNA Primers; D005420:Flavoproteins; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase; C082502:petH protein, Bacteria