Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

Photosynth Res 2015 Aug 20;125(1-2):321-8. Epub 2015 Feb 20.

Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa, 920-1192, Japan,

Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group.

Download full-text PDF

Source
http://dx.doi.org/10.1007/s11120-015-0099-8DOI Listing
August 2015
14 Reads

Publication Analysis

Top Keywords

wild type
16
prosthetic group
12
bacillus subtilis
8
presence nadph
8
tyr50 gly
8
y50g y50s
8
isoalloxazine ring
8
diaphorase assay
8
replacement tyr50
8
y50w mutant
8
nadph-thioredoxin reductase
8
bacterial nadph-thioredoxin
8
types flavoproteins
4
three mutants
4
flavoproteins including
4
quenched three
4
including plant-type
4
bsfnr blue
4
fnr flavodoxin
4
fad transition
4

References

(Supplied by CrossRef)

A Aliverti et al.
1999

A Aliverti et al.
Arch Biochem Biophys 2008

AK Arakaki et al.
J Biol Chem 2001

S Baroni et al.
Biochemistry 2012

CJ Batie et al.
J Biol Chem 1984

EA Ceccarelli et al.
Biochim Biophys Acta 2004

CC Correll et al.
Protein Sci 1993

O Dym et al.
Protein Sci 2001

KM Ewen et al.
Biochim Biophys Acta 2011

AJ Green et al.
J Inorg Biochem 2003

PF Heelis et al.
Chem Soc Rev 1982

Similar Publications