Studies on the active centre of Rhodotorula gracilis D-amino acid oxidase and comparison with pig kidney enzyme

• Published in: Biochemical journal 1992-09, Vol.286 (2), p.389-394
• Main Authors: POLLEGIONI, L, GHISLA, S, PILONE, M. S
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; D-Amino-Acid Oxidase - metabolism; Enzymes and enzyme inhibitors; Flavin-Adenine Dinucleotide - analogs & derivatives; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Kidney - enzymology; Oxidoreductases; Rhodotorula - enzymology; Rhodotorula gracilis; Swine
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj2860389

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.