Monomer formation and functioning of p hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide-water mixtures

E.V. Kudryashova, A.J.W.G. Visser, W.J.H. van Berkel

Research output: Contribution to journalArticleAcademicpeer-review

8 Citations (Scopus)

Abstract

It has previously been postulated that the dimeric form of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH) is important for catalysis. Here it is demonstrated that the monomeric form of PHBH is active. In a water/AOT/isooctane reverse micellar system, the function of the monomeric and dimeric forms of PHBH could be observed separately by varying the size of the micelles. A considerable decrease in the KM value for p-hydroxybenzoate (POHB) was found for monomeric PHBH, accompanied by a 1.5-fold decrease in enzymatic activity. The same tendency was observed when monomers of PHBH were formed by adding DMSO to the buffer. The FAD in PHBH and PHBH labeled with the fluorescence dye Alexa488 was investigated by time-resolved fluorescence anisotropy to observe monomer formation in water/DMSO mixtures. Monomer formation of PHBH occurred gradually with increasing DMSO content in the mixture. Pure PHBH monomers were detected at DMSO concentrations of 30 % (v/v) and higher.
Original languageEnglish
Pages (from-to)413-419
JournalChemBioChem
Volume9
Issue number3
DOIs
Publication statusPublished - 2008

Keywords

  • time-resolved fluorescence
  • flavin adenine-dinucleotide
  • pseudomonas-fluorescens
  • alpha-chymotrypsin
  • catalytic activity
  • crystal-structure
  • escherichia-coli
  • mobile flavin
  • aerosol ot
  • wild-type

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