Functional and mechanistic characterization of an atypical
flavin reductase encoded by the pden_5119 gene in Paracoccus
denitrificans

J 2019

Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans

SEDLÁČEK, Vojtěch and Igor KUČERA

Basic information

Original name

Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans

Authors

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

Edition

Molecular Microbiology, HOBOKEN, WILEY, 2019, 0950-382X

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 3.418

RIV identification code

RIV/00216224:14310/19:00107524

Organization unit

Faculty of Science

DOI

http://dx.doi.org/10.1111/mmi.14260

UT WoS

000474705900011

Keywords in English

KINETIC MECHANISM; REDUCED FLAVIN; OXIDATIVE STRESS; SUPEROXIDE ANION; NADH OXIDASE

Abstract

V originále

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of k(cat) demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.

Links

GA16-18476S, research and development project

Name: Oxidační stres u denitrifikačních baktérií: objasnění funkce zúčastněných proteinů a možných dopadů na životní prostředí

Investor: Czech Science Foundation

LM2011020, research and development project

Name: CEITEC ? open access

Investor: Ministry of Education, Youth and Sports of the CR

Citovat

SEDLÁČEK, Vojtěch and Igor KUČERA. Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans. Molecular Microbiology. HOBOKEN: WILEY, 2019, vol. 112, No 1, p. 166-183. ISSN 0950-382X. Available from: https://dx.doi.org/10.1111/mmi.14260.

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   author = {Sedláček, Vojtěch and Kučera, Igor},
   article_location = {HOBOKEN},
   article_number = {1},
   doi = {http://dx.doi.org/10.1111/mmi.14260},
   keywords = {KINETIC MECHANISM; REDUCED FLAVIN; OXIDATIVE STRESS; SUPEROXIDE ANION; NADH OXIDASE},
   language = {eng},
   issn = {0950-382X},
   journal = {Molecular Microbiology},
   title = {Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans},
   url = {http://dx.doi.org/10.1111/mmi.14260},
   volume = {112},
   year = {2019}
}

TY  - JOUR
ID  - 1547016
AU  - Sedláček, Vojtěch - Kučera, Igor
PY  - 2019
TI  - Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden_5119 gene in Paracoccus denitrificans
JF  - Molecular Microbiology
VL  - 112
IS  - 1
SP  - 166-183
EP  - 166-183
PB  - WILEY
SN  - 0950382X
KW  - KINETIC MECHANISM
KW  - REDUCED FLAVIN
KW  - OXIDATIVE STRESS
KW  - SUPEROXIDE ANION
KW  - NADH OXIDASE
UR  - http://dx.doi.org/10.1111/mmi.14260
L2  - http://dx.doi.org/10.1111/mmi.14260
N2  - Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of k(cat) demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.
ER  -

SEDLÁČEK, Vojtěch and Igor KUČERA. Functional and mechanistic characterization of an atypical flavin reductase encoded by the pden\_{}5119 gene in Paracoccus denitrificans. \textit{Molecular Microbiology}. HOBOKEN: WILEY, 2019, vol.~112, No~1, p.~166-183. ISSN~0950-382X. Available from: https://dx.doi.org/10.1111/mmi.14260.

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