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Journal of Biochemistry Advance Access originally published online on January 3, 2009
Journal of Biochemistry 2009 145(4):481-491; doi:10.1093/jb/mvn185
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

In Vitro and In Vivo Interactions of Ferredoxin-NADP+ Reductases in Pseudomonas putida

Jinki Yeom1, Che Ok Jeon2, Eugene L. Madsen3 and Woojun Park1,*

1Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-075; 2Department of Life Science, Chung-Ang University, Seoul 156-756, Korea; and 3Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA

*To whom correspondence should be addressed: Tel: +82-2-3290-3067, Fax: +82-2-953-0737, E-mail: wpark{at}korea.ac.kr

Received November 26, 2008; Accepted December 26, 2008


  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.

Key Words: far-western blot, homology modelling, oxidative stress, protein-protein interaction, Pseudomonas putida KT2440

Abbreviations: Fpr, Ferredoxin-NADP+ reductase; Fd, [2Fe-2S] Ferredoxin; 4Fd, [4Fe-4S] Ferredoxin; Fld, Flavodoxin; FdxA, [4Fe-4S] Ferredoxin of P. Putida similar to FdI of A. vinelandii; DPIP, 2,6-dichlorophenolindophenol; NBT, Nitroblue tetrazolium


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