Protein Engineering vol. 16 no. 12 pp. 1063-1069, 2003
© 2003 Oxford University Press
Dissection of the structural determinants involved in formation of the dimeric form of D-amino acid oxidase from Rhodotorula gracilis: role of the size of the ßF5–ßF6 loop
Department of Structural and Functional Biology, University of Insubria, Via J. H. Dunant, 3, 21100 Varese, Italy
1 To whom correspondence should be addressed. e-mail: luciano.piubelli{at}uninsubria.it
The role of the long loop connecting ß-strands F5 and F6 (21 amino acids, Pro302–Leu–Asp–Arg–Thr–Lys–Ser–Pro–Leu–Ser–Leu–Gly–Arg–Gly–Ser–Ala–Arg–Ala–Ala–Lys–Glu322) present in Rhodotorula gracilis D-amino acid oxidase (RgDAAO) was investigated by site-directed mutagenesis. This loop was proposed to play an important role in the ‘head-to-tail’ monomer–monomer interaction of this dimeric flavoenzyme: in particular, by means of electrostatic interactions between positively charged residues of the ßF5–ßF6 loop of one monomer and negatively charged residues belonging to the 
-helices I3' and I3'' of the other monomer. We produced a mutant of RgDAAO (namely, DAAO–
LOOP2), in which only minor structural perturbations were introduced (only five amino acids were deleted; new sequence of the ßF5–ßF6 loop is Pro302–Leu–Asp–Arg–Thr–Leu–Gly–Arg–Gly–Ser–Ala–Arg–Ala–Ala–Lys–Glu317), and the charge of the ßF5–ßF6 loop not modified. The LOOP2 mutant is monomeric, has a weaker binding with the FAD cofactor, a decrease of the kinetic efficiency, and slight modifications in its spectral properties. The short version of the loop does not allow a correct monomer–monomer interaction, and its presence in the monomeric DAAO is a destabilizing structural element since the LOOP2 mutant is highly susceptible to proteolysis. These results, confirming the role of this loop in the subunits interaction and thus in stabilization of the sole dimeric form of RgDAAO, put forward the evidence that even a short deletion of the loop generates a consistent variation of the enzyme structure–function properties.
Received July 9, 2003; revised September 28, 2003; accepted October 21, 2003
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