Vibrio harveyi NADPH: FMN oxidoreductase: Preparation and characterization of the apoenzyme and monomer-dimer equilibrium

Mengyao Liu, Benfang Lie, Qizhu Ding, James Lee, Shiao Chun Tu

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with ≤80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 μM at 23°C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme, 2ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4°C by equilibrium ultracentrifugation to undergo dimerizationwith dissociation constants of 1.8 and 3.3 μM, respectively.

Original languageEnglish (US)
Pages (from-to)89-95
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume337
Issue number1
DOIs
StatePublished - Jan 1 1997

Fingerprint

FMN Reductase
Apoenzymes
Holoenzymes
Vibrio
Flavin Mononucleotide
NADP
Dimers
Monomers
Chromatography
Flavins
Flavin-Adenine Dinucleotide
Apoproteins
Riboflavin
Ultracentrifugation
Molecular sieves
Substrates
Enzymes
Gel Chromatography
Quenching
Molecular Weight

Keywords

  • apoenzyme
  • dimerization
  • flavin reductase
  • NADPH:FMN oxidoreductase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Vibrio harveyi NADPH : FMN oxidoreductase: Preparation and characterization of the apoenzyme and monomer-dimer equilibrium. / Liu, Mengyao; Lie, Benfang; Ding, Qizhu; Lee, James; Tu, Shiao Chun.

In: Archives of Biochemistry and Biophysics, Vol. 337, No. 1, 01.01.1997, p. 89-95.

Research output: Contribution to journalArticle

Liu, Mengyao ; Lie, Benfang ; Ding, Qizhu ; Lee, James ; Tu, Shiao Chun. / Vibrio harveyi NADPH : FMN oxidoreductase: Preparation and characterization of the apoenzyme and monomer-dimer equilibrium. In: Archives of Biochemistry and Biophysics. 1997 ; Vol. 337, No. 1. pp. 89-95.
@article{f60c89bdf0a04471bdbfb1ee056cf1f2,
title = "Vibrio harveyi NADPH: FMN oxidoreductase: Preparation and characterization of the apoenzyme and monomer-dimer equilibrium",
abstract = "A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with ≤80{\%} yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 μM at 23°C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92{\%} of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme, 2ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4°C by equilibrium ultracentrifugation to undergo dimerizationwith dissociation constants of 1.8 and 3.3 μM, respectively.",
keywords = "apoenzyme, dimerization, flavin reductase, NADPH:FMN oxidoreductase",
author = "Mengyao Liu and Benfang Lie and Qizhu Ding and James Lee and Tu, {Shiao Chun}",
year = "1997",
month = "1",
day = "1",
doi = "10.1006/abbi.1996.9746",
language = "English (US)",
volume = "337",
pages = "89--95",
journal = "Archives of Biochemistry and Biophysics",
issn = "0003-9861",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Vibrio harveyi NADPH

T2 - FMN oxidoreductase: Preparation and characterization of the apoenzyme and monomer-dimer equilibrium

AU - Liu, Mengyao

AU - Lie, Benfang

AU - Ding, Qizhu

AU - Lee, James

AU - Tu, Shiao Chun

PY - 1997/1/1

Y1 - 1997/1/1

N2 - A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with ≤80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 μM at 23°C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme, 2ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4°C by equilibrium ultracentrifugation to undergo dimerizationwith dissociation constants of 1.8 and 3.3 μM, respectively.

AB - A rapid chromatography method was developed for the preparation of apoenzyme of Vibrio harveyi NADPH:FMN oxidoreductase with ≤80% yields. The apoenzyme bound one FMN per enzyme monomer with a dissociation constant of 0.2 μM at 23°C. The reconstituted holoenzyme was catalytically as active as the native enzyme. FMN binding resulted in 87 and 92% of quenching of protein and flavin fluorescence, respectively, indicating a conformational difference between the apoprotein and the holoenzyme. Neither riboflavin nor FAD showed any appreciable binding to the cofactor site of the apoenzyme but both flavins were active substrates for the FMN-containing holoenzyme, 2ThioFMN bound to the cofactor site of the apoenzyme with an affinity similar to that for FMN binding. The holoenzyme reconstituted with 2-thioFMN showed a 509-nm absorption peak, which represents a 19-nm red shift from the corresponding peak of the free flavin, and was catalytically active in using either FMN or 2thioFMN as a substrate. The holoenzyme showed a concentration dependence in molecular sieve chromatography corresponding to higher apparent molecular weights at higher concentrations. Both the holoenzyme and the apoenzyme was shown at 4°C by equilibrium ultracentrifugation to undergo dimerizationwith dissociation constants of 1.8 and 3.3 μM, respectively.

KW - apoenzyme

KW - dimerization

KW - flavin reductase

KW - NADPH:FMN oxidoreductase

UR - http://www.scopus.com/inward/record.url?scp=0030902764&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030902764&partnerID=8YFLogxK

U2 - 10.1006/abbi.1996.9746

DO - 10.1006/abbi.1996.9746

M3 - Article

C2 - 8990272

AN - SCOPUS:0030902764

VL - 337

SP - 89

EP - 95

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

IS - 1

ER -