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Monomer Formation and Function of p ‐Hydroxybenzoate Hydroxylase in Reverse Micelles and in Dimethylsulfoxide/Water Mixtures
Author(s) -
Kudryashova Elena V.,
Visser Antonie J. W. G.,
van Berkel Willem J. H.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700267
Subject(s) - monomer , flavoprotein , chemistry , micelle , biochemistry , polymer , enzyme , organic chemistry , aqueous solution
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 K M 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.