Purification and characterization of ferric reductases from
Paracoccus denitrificans

D 2002

Purification and characterization of ferric reductases from Paracoccus denitrificans

TESAŘÍK, Radek, Jiří MAZOCH, Jaroslav TURÁNEK a Igor KUČERA

Základní údaje

Originální název

Purification and characterization of ferric reductases from Paracoccus denitrificans

Autoři

TESAŘÍK, Radek (203 Česká republika), Jiří MAZOCH (203 Česká republika), Jaroslav TURÁNEK (203 Česká republika) a Igor KUČERA (203 Česká republika, garant)

Vydání

Bratislava, XVIII. BIOCHEMICKÝ ZJAZD, s. 150-150, 2002

Nakladatel

Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Slovensko

Utajení

není předmětem státního či obchodního tajemství

Kód RIV

RIV/00216224:14310/02:00006390

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

ferric reductase; Paracoccus denitrificans; purification

Štítky

ferric reductase, Paracoccus denitrificans, purification

Změněno: 13. 5. 2003 09:07, Mgr. Jiří Mazoch, Ph.D.

Anotace

V originále

Iron is a component of a number of biological systems, e.g. electron transport chains, cofactors of enzymes, regulatory proteins etc. It occurs in ferric form in environment, while organisms require ferrous iron. Enzymes catalysing the reduction of ferric complexes are widely spread within bacterial kingdom. Some of these enzymes are associated with cytoplasmic membrane, some are present in soluble form in cytoplasm or periplasm, some are excreted into extracellular medium. In our work, cellular fractions of Paracoccus denitrificans were tested for their activity towards reduction of various ferric complexes with NADH as an electron donor. We found the most of the ferric reductase activity in cytosolic fraction. Fe(III)-nitrilotriacetate was chosen as the best artificial substrate for activity measurements. Two proteins responsible for the activity was identified. Both enzymes were purified to homogeneity by combination of FPLC ion-exchange chromatography and chromatofocusing, and characterised with respect to kinetics of enzyme reaction. One of these enzymes (denoted FerB) does not need FMN or other flavins for its catalytic function. It differs in this aspect from other known ferric reductases which are considered to be in fact flavin reductases (Fontecave et al., 1994). Both NADH and NADPH serve as electron donors for FerB whereas FerA uses NADH exclusively. Mr of native enzymes were estimated (GPC) to be about 25 kDa and 55 kDa for FerA (pI 6.9) and FerB (pI 5.5), respectively. N-terminal sequences of FerA, FerB were determined and database searches revealed that the sequence of FerB is homologous to chromate reductase of Pseudomonas putida. Indeed, FerB has chromate reductase activity.

Návaznosti

GA203/01/1589, projekt VaV

Název: Mechanizmy regulace respiračních systémů denitrifikačních bakterií faktory prostředí

Investor: Grantová agentura ČR, Mechanizmy regulace respiračních systémů denitrifikačních bakterií faktory prostředí

Citovat

TESAŘÍK, Radek, Jiří MAZOCH, Jaroslav TURÁNEK a Igor KUČERA. Purification and characterization of ferric reductases from Paracoccus denitrificans. In XVIII. BIOCHEMICKÝ ZJAZD. Bratislava: Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV, 2002, s. 150.

@inproceedings{405236,
   author = {Tesařík, Radek and Mazoch, Jiří and Turánek, Jaroslav and Kučera, Igor},
   address = {Bratislava},
   booktitle = {XVIII. BIOCHEMICKÝ ZJAZD},
   keywords = {ferric reductase; Paracoccus denitrificans; purification},
   language = {eng},
   location = {Bratislava},
   pages = {150-150},
   publisher = {Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV},
   title = {Purification and characterization of ferric reductases from Paracoccus denitrificans},
   year = {2002}
}

TY  - JOUR
ID  - 405236
AU  - Tesařík, Radek - Mazoch, Jiří - Turánek, Jaroslav - Kučera, Igor
PY  - 2002
TI  - Purification and characterization of ferric reductases from Paracoccus denitrificans
PB  - Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV
CY  - Bratislava
KW  - ferric reductase
KW  - Paracoccus denitrificans
KW  - purification
N2  - Iron is a component of a number of biological systems, e.g. electron transport chains, cofactors of enzymes, regulatory proteins etc. It occurs in ferric form in environment, while organisms require ferrous iron. Enzymes catalysing the reduction of ferric complexes are widely spread within bacterial kingdom. Some of these enzymes are associated with cytoplasmic membrane, some are present in soluble form in cytoplasm or periplasm, some are excreted into extracellular medium. In our work, cellular fractions of Paracoccus denitrificans were tested for their activity towards reduction of various ferric complexes with NADH as an electron donor. We found the most of the ferric reductase activity in cytosolic fraction. Fe(III)-nitrilotriacetate was chosen as the best artificial substrate for activity measurements. Two proteins responsible for the activity was identified. Both enzymes were purified to homogeneity by combination of FPLC ion-exchange chromatography and chromatofocusing, and characterised with respect to kinetics of enzyme reaction. One of these enzymes (denoted FerB) does not need FMN or other flavins for its catalytic function. It differs in this aspect from other known ferric reductases which are considered to be in fact flavin reductases (Fontecave et al., 1994). Both NADH and NADPH serve as electron donors for FerB whereas FerA uses NADH exclusively. Mr of native enzymes were estimated (GPC) to be about 25 kDa and 55 kDa for FerA (pI 6.9) and FerB (pI 5.5), respectively. N-terminal sequences of FerA, FerB were determined and database searches revealed that the sequence of FerB is homologous to chromate reductase of Pseudomonas putida. Indeed, FerB has chromate reductase activity.
ER  -

TESAŘÍK, Radek, Jiří MAZOCH, Jaroslav TURÁNEK a Igor KUČERA. Purification and characterization of ferric reductases from Paracoccus denitrificans. In \textit{XVIII. BIOCHEMICKÝ ZJAZD}. Bratislava: Slovenská spoločnosť pre biochémiu a molekulárnu biológiu pri SAV, 2002, s.~150.

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