Site-directed mutagenesis in the active site of Rhodotorula gracilis D-amino acid oxidase

1999 ◽  
Vol 27 (1) ◽  
pp. A38-A38
Author(s):  
M.S. Pilone ◽  
G. Molla ◽  
C. Harris ◽  
D. Porrini ◽  
C. Vegezzi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

scholarly journals Limited proteolysis and site-directed mutagenesis reveal the origin of microheterogeneity in Rhodotorula gracilis D-amino acid oxidase

1998 ◽  
Vol 330 (2) ◽  
pp. 615-621 ◽  
Author(s):  
Stefano CAMPANER ◽  
Loredano POLLEGIONI ◽  
D. Brian ROSS ◽  
S. Mirella PILONE
Keyword(s):  
Amino Acid ◽  
Isoelectric Focusing ◽  
Limited Proteolysis ◽  
Single Band ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Oxidase ◽  
Wild Type ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

When analysed by isoelectric focusing, D-amino acid oxidase from the yeast Rhodotorula gracilis normally consists of three molecular isoforms (pI 7.8, 7.4 and 7.2, respectively) all with the same N-terminal sequence. However, only a single band of pI 7.8 is detected with the recombinant wild-type protein expressed in E. coli. To determine whether the molecular basis of this heterogeneity is due to proteolysed forms of the protein, we treated R. gracilisd-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of ≈ 34 kDa (Met1-Leu312) and one of ≈ 5 kDa (Ala319-Arg364 with chymotrypsin or Ala319-Ala362 with thermolysin). On the other hand, treatment with endoproteinase Glu-C gave a dimeric holoenzyme lacking the C-terminal SKL tripeptide. This cleavage of Glu365-Ser366 peptide bond caused the disappearance of the three isoelectric bands and a single homogeneous band (pI 7.2) appeared. To study this protein form, we used site-directed mutagenesis to produce a mutant form of R. gracilisD-amino acid oxidase lacking the SKL C-terminal tripeptide (which is the targeting sequence PTS1 for peroxisomal proteins). As expected, the SKL-deleted mutant gave a single band (pI 7.2) in isoelectric focusing. The three-band pattern of native yeast enzyme was generated by in vitro experiments using an equimolar mixture of the wild-type (pI 7.8) and the SKL-deleted recombinant (pI 7.2) DAAOs. The microheterogeneity of yeast DAAO thus stems from the association of two polypeptide chains differing in the C-terminal tripeptide, giving three different holoenzyme dimers.

scholarly journals Role of tyrosine 238 in the active site of Rhodotorula gracilis d -amino acid oxidase

2002 ◽  
Vol 269 (19) ◽  
pp. 4762-4771 ◽  
Author(s):  
Angelo Boselli ◽  
Silvia Sacchi ◽  
Viviana Job ◽  
Mirella S. Pilone ◽  
Loredano Pollegioni
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

Investigating the role of active site residues of Rhodotorula gracilis d-amino acid oxidase on its substrate specificity

Biochimie ◽  
2007 ◽  
Vol 89 (3) ◽  
pp. 360-368 ◽  
Author(s):  
Angelo Boselli ◽  
Luciano Piubelli ◽  
Gianluca Molla ◽  
Mirella S. Pilone ◽  
Loredano Pollegioni ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Active Site ◽  
Acid Oxidase ◽  
Active Site Residues ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

Site-directed mutagenesis of D-amino acid oxidase from yeast Trigonopsis variabilis

2010 ◽  
Vol 150 ◽  
pp. 442-442 ◽  
Author(s):  
N.V. Cherskova ◽  
S.V. Khoronenkova ◽  
M.A. Panteleev ◽  
V.I. Tishkov
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Trigonopsis Variabilis

scholarly journals On the mechanism of Rhodotorula gracilis d-amino acid oxidase: role of the active site serine 335

2004 ◽  
Vol 1702 (1) ◽  
pp. 19-32 ◽  
Author(s):  
Angelo Boselli ◽  
Luciano Piubelli ◽  
Gianluca Molla ◽  
Silvia Sacchi ◽  
Mirella S. Pilone ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis

Effect of hydrogen peroxide on d-amino acid oxidase from Rhodotorula gracilis

2000 ◽  
Vol 27 (3-5) ◽  
pp. 234-239 ◽  
Author(s):  
Isabel de la Mata ◽  
Fernando Ramón ◽  
Virginia Obregón ◽  
Ma Pilar Castillón ◽  
Carmen Acebal
Keyword(s):  
Hydrogen Peroxide ◽  
Amino Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Rhodotorula Gracilis
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