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

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. gracilis D-amino acid oxidase with various proteases. Limited proteolysis by chymotrypsin and thermolysin produced truncated and nicked monomeric holoenzymes containing two polypeptides of approximately 34 kDa (Met1-Leu312) and one of approximately 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. gracilis D-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.