Studies on the active centre of Rhodotorula gracilis D-amino acid oxidase and comparison with pig kidney enzyme.
D-Amino acid oxidase (EC 1.4.3.3) from Rhodotorula gracilis has been reconstituted with 8-chloro-, 8-mercapto-, 6-hydroxy-, 2-thio-, 5-deaza- and 1-deaza-FAD, and the properties of the resulting complexes have been studied and compared with those of the correspondingly modified pig kidney D-amino acid oxidases. Binding appears to be tight for most analogues, at least as tight as for native FAD (approximately 10(-8) M). 8-Mercapto- and 6-hydroxy-FAD bind in their para- and ortho-quinoid forms respectively to yeast D-amino acid oxidase, inferring the presence of a positive charge near the flavin N(1) position, as in the case of the mammalian enzyme. On the other hand, important differences in active-site microenvironment emerge: solvent accessibility to flavin position 8 is drastically restricted in yeast D-amino acid oxidase as indicated by the unreactivity of 8-chloro- and 8-mercapto-FAD enzyme with thiolates and alkylating agents. Significantly different microenvironments are also likely to occur around the flavin positions N(1)-C(2) = 0, N(3)-H and N(5). This is deduced from the differences in interaction of the two proteins with 1-deaza-FAD, 5-deaza-FAD and 2-thio-FAD and from the properties of the respective complexes. The same re-side flavin stereospecificity as shown by the mammalian enzyme was determined for the yeast enzyme using 8-hydroxy-5-deaza-FAD. Thus we can deduce the presence of a similar pattern of functional groups at the active centres of the two enzymes, while the fine tuning of specificity and regulation correlate with environmental differences at specific flavin loci.
Department of General Physiology and Biochemistry, University of Milano, Italy.
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D000818:Animals; D001665:Binding Sites; D003605:D-Amino-Acid Oxidase / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D012248:Rhodotorula / Q000201:enzymology*; D013552:Swine
D005182:Flavin-Adenine Dinucleotide; D003605:D-Amino-Acid Oxidase