Studies on the structural and functional aspects of Rhodotorula gracilis D-amino acid oxidase by limited trypsinolysis.
The structure-function relationships of purified Rhodotorula gracilis D-amino acid oxidase (in its holo-, apo- and holo-enzyme-benzoate complex forms) was analysed by digestion with trypsin. In all cases trypsin cleaves this 80 kDa dimeric enzyme at the C-terminal region, since the peptide bonds sensitive to proteinase attack are clustered in this region. Digestion of native enzyme with trypsin produced a nicked and truncated form of 38.3 kDa containing two polypeptides of 34 and 5 kDa starting from Met1 and Ala319 respectively, and with detachment of the Thr306-Arg318 and Glu365-Leu368 peptides. Our results show that this 'core', folded into a compact structure, is catalytically competent. The acquisition of this nicked form was marked by a shift from a dimeric to a monomeric active enzyme, a result never previously obtained. The deleted sequences, Thr306-Arg318 and Glu365-Leu368, are essential for the monomer-monomer interaction, and, in particular, the region encompassing Thr306-Arg318 should play an essential role in the dimerization process. interestingly, the Ser308-Lys321 sequence present in the lost peptide corresponds to a sequence not present in other known D-amino acid oxidases [Faotto, Pollegioni, Ceciliani, Ronchi and Pilone (1995) Biotechnol. Lett. 17, 193-198]. A role of the cleaved-off region for the thermostabilization of the enzyme is also discussed.
Pollegioni
L
L
Centro Interuniversitario per lo Studio delle Macromolecole Informazionali, University of Milano, Italy.
Ceciliani
F
F
Curti
B
B
Ronchi
S
S
Pilone
M S
MS
D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D001565:Benzoates; D002151:Calorimetry; D002851:Chromatography, High Pressure Liquid; D003605:D-Amino-Acid Oxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D012248:Rhodotorula / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D014357:Trypsin
D001051:Apoenzymes; D001565:Benzoates; D010446:Peptide Fragments; D003605:D-Amino-Acid Oxidase; D014357:Trypsin