Abstract
Ferredoxin-NADP+ reductase (Fpr) is known to control NADP +/NADPH pool in proteobacteria. There is only one fpr gene present in most proteobacteria, but Pseudomonas putida has two Fprs (FprA and FprB). We elucidated the functional relationships between the two types of Fpr and their electron transport partners [ferredoxin (Fd) and flavodoxin (Fld)] by cloning, expressing and preparing these proteins in various combinations and assessing their properties in vitro and in vivo using biochemical assays, the Far-western analysis, the yeast two-hybrid assay and structural molecular modelling. Both of the Fprs have a lower Km value for NADPH than for NADH in the diaphorase assays. With NADH as electron donor, FprB also has a high specific constant (kcat/Km) in the diaphorase assay. The catalytic efficiency of FprA is higher when Fld is present as its redox partner, compared to the kinetics observed with other electron transport partners in a NADPH-dependent cytochrome c reduction assay. The highest specific constant (kcat/Km) of FprB was observed in the presence of FdA. FprB's Km value and catalytic activity (kcat) with NADH were significant in cytochrome c reduction assays. Strong kinetic interactions of Fprs with their redox partners were also demonstrated by homology modelling, the Far-western analysis and the in vivo yeast two-hybrid system. This study demonstrates for the first time that Fprs in P. putida function as diaphorase, Fd/Fld reductases and determines their preferred redox partner in vivo and in vitro.
| Original language | English |
|---|---|
| Pages (from-to) | 481-491 |
| Number of pages | 11 |
| Journal | Journal of Biochemistry |
| Volume | 145 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2009 Apr 1 |
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Keywords
- Far-western blot
- Homology modelling
- Oxidative stress
- Protein-protein interaction
- Pseudomonas putida KT2440
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
In vitro and in vivo interactions of ferredoxin-NADP+ reductases in Pseudomonas putida. / Yeom, Jinki; Jeon, Che Ok; Madsen, Eugene L.; Park, Woojun.
In: Journal of Biochemistry, Vol. 145, No. 4, 01.04.2009, p. 481-491.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - In vitro and in vivo interactions of ferredoxin-NADP+ reductases in Pseudomonas putida
AU - Yeom, Jinki
AU - Jeon, Che Ok
AU - Madsen, Eugene L.
AU - Park, Woojun
PY - 2009/4/1
Y1 - 2009/4/1
N2 - Ferredoxin-NADP+ reductase (Fpr) is known to control NADP +/NADPH pool in proteobacteria. There is only one fpr gene present in most proteobacteria, but Pseudomonas putida has two Fprs (FprA and FprB). We elucidated the functional relationships between the two types of Fpr and their electron transport partners [ferredoxin (Fd) and flavodoxin (Fld)] by cloning, expressing and preparing these proteins in various combinations and assessing their properties in vitro and in vivo using biochemical assays, the Far-western analysis, the yeast two-hybrid assay and structural molecular modelling. Both of the Fprs have a lower Km value for NADPH than for NADH in the diaphorase assays. With NADH as electron donor, FprB also has a high specific constant (kcat/Km) in the diaphorase assay. The catalytic efficiency of FprA is higher when Fld is present as its redox partner, compared to the kinetics observed with other electron transport partners in a NADPH-dependent cytochrome c reduction assay. The highest specific constant (kcat/Km) of FprB was observed in the presence of FdA. FprB's Km value and catalytic activity (kcat) with NADH were significant in cytochrome c reduction assays. Strong kinetic interactions of Fprs with their redox partners were also demonstrated by homology modelling, the Far-western analysis and the in vivo yeast two-hybrid system. This study demonstrates for the first time that Fprs in P. putida function as diaphorase, Fd/Fld reductases and determines their preferred redox partner in vivo and in vitro.
AB - Ferredoxin-NADP+ reductase (Fpr) is known to control NADP +/NADPH pool in proteobacteria. There is only one fpr gene present in most proteobacteria, but Pseudomonas putida has two Fprs (FprA and FprB). We elucidated the functional relationships between the two types of Fpr and their electron transport partners [ferredoxin (Fd) and flavodoxin (Fld)] by cloning, expressing and preparing these proteins in various combinations and assessing their properties in vitro and in vivo using biochemical assays, the Far-western analysis, the yeast two-hybrid assay and structural molecular modelling. Both of the Fprs have a lower Km value for NADPH than for NADH in the diaphorase assays. With NADH as electron donor, FprB also has a high specific constant (kcat/Km) in the diaphorase assay. The catalytic efficiency of FprA is higher when Fld is present as its redox partner, compared to the kinetics observed with other electron transport partners in a NADPH-dependent cytochrome c reduction assay. The highest specific constant (kcat/Km) of FprB was observed in the presence of FdA. FprB's Km value and catalytic activity (kcat) with NADH were significant in cytochrome c reduction assays. Strong kinetic interactions of Fprs with their redox partners were also demonstrated by homology modelling, the Far-western analysis and the in vivo yeast two-hybrid system. This study demonstrates for the first time that Fprs in P. putida function as diaphorase, Fd/Fld reductases and determines their preferred redox partner in vivo and in vitro.
KW - Far-western blot
KW - Homology modelling
KW - Oxidative stress
KW - Protein-protein interaction
KW - Pseudomonas putida KT2440
UR - http://www.scopus.com/inward/record.url?scp=67649654270&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=67649654270&partnerID=8YFLogxK
U2 - 10.1093/jb/mvn185
DO - 10.1093/jb/mvn185
M3 - Article
C2 - 19122206
AN - SCOPUS:67649654270
VL - 145
SP - 481
EP - 491
JO - Journal of Biochemistry
JF - Journal of Biochemistry
SN - 0021-924X
IS - 4
ER -