Biochemical properties and crystal structure of the flavin
reductase FerA from Paracoccus denitrificans

J 2016

Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans

SEDLÁČEK, Vojtěch, Tomáš KLUMPLER, Jaromír MAREK and Igor KUČERA

Basic information

Original name

Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans

Authors

SEDLÁČEK, Vojtěch (203 Czech Republic, belonging to the institution), Tomáš KLUMPLER (203 Czech Republic, belonging to the institution), Jaromír MAREK (203 Czech Republic, belonging to the institution) and Igor KUČERA (203 Czech Republic, guarantor, belonging to the institution)

Edition

Microbiological Research, ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2016, 0944-5013

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Germany

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 3.037

RIV identification code

RIV/00216224:14310/16:00088000

Organization unit

Faculty of Science

DOI

http://dx.doi.org/10.1016/j.micres.2016.04.006

UT WoS

000378963100002

Keywords in English

FerA; FMN; FAD; flavin reductase family; oxidative stress; Paracoccus denitrificans

Abstract

V originále

The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel b-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.

Links

GAP503/12/0369, research and development project

Name: Nové flavin-dependentní enzymy Paracoccus denitrificans: reakční mechanismy, metabolické funkce a úloha v buněčném oxidačním stresu

Investor: Czech Science Foundation

Citovat

SEDLÁČEK, Vojtěch, Tomáš KLUMPLER, Jaromír MAREK and Igor KUČERA. Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans. Microbiological Research. ELSEVIER GMBH, URBAN & FISCHER VERLAG, 2016, vol. 188, No 1, p. 9-22. ISSN 0944-5013. Available from: https://dx.doi.org/10.1016/j.micres.2016.04.006.

@article{1348292,
   author = {Sedláček, Vojtěch and Klumpler, Tomáš and Marek, Jaromír and Kučera, Igor},
   article_number = {1},
   doi = {http://dx.doi.org/10.1016/j.micres.2016.04.006},
   keywords = {FerA; FMN; FAD; flavin reductase family; oxidative stress; Paracoccus denitrificans},
   language = {eng},
   issn = {0944-5013},
   journal = {Microbiological Research},
   title = {Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans},
   url = {http://www.sciencedirect.com/science/article/pii/S0944501316301549},
   volume = {188},
   year = {2016}
}

TY  - JOUR
ID  - 1348292
AU  - Sedláček, Vojtěch - Klumpler, Tomáš - Marek, Jaromír - Kučera, Igor
PY  - 2016
TI  - Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans
JF  - Microbiological Research
VL  - 188
IS  - 1
SP  - 9-22
EP  - 9-22
PB  - ELSEVIER GMBH, URBAN & FISCHER VERLAG
SN  - 09445013
KW  - FerA
KW  - FMN
KW  - FAD
KW  - flavin reductase family
KW  - oxidative stress
KW  - Paracoccus denitrificans
UR  - http://www.sciencedirect.com/science/article/pii/S0944501316301549
L2  - http://www.sciencedirect.com/science/article/pii/S0944501316301549
N2  - The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel b-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H:acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.
ER  -

SEDLÁČEK, Vojtěch, Tomáš KLUMPLER, Jaromír MAREK and Igor KUČERA. Biochemical properties and crystal structure of the flavin reductase FerA from Paracoccus denitrificans. \textit{Microbiological Research}. ELSEVIER GMBH, URBAN \&{} FISCHER VERLAG, 2016, vol.~188, No~1, p.~9-22. ISSN~0944-5013. Available from: https://dx.doi.org/10.1016/j.micres.2016.04.006.

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