PURIFICATION AND PARTIAL CHARACTERIZATION OF A GLUTAMYL-TRANSFER RNA-SYNTHETASE FROM THE UNICELLULAR GREEN-ALGA SCENEDESMUS-OBLIQUUS, MUTANT C-2A'

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNA(Glu) by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482-1484). The synthetase from the yellow pigment mutant C-2A' of the unicellular green alga Sc enedesmus obliquus was purified by sequential column chromatography on Sephacryl S300, Blue Sepharose, phosphocellulose P11 and by fast prot ein liquid chromatography (FPLC) on Mono Q. After denaturing sodium do decylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, provi ng the apparent homogeneity of the glutamyl-tRNA synthetase. A molecul ar mass of 105+/-10 kDa was determined for the native protein by chrom atography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The puri fied protein is active within a pH range from 7.0 to 9.0 with a maximu m activity at pH 8.0. Kinetics for the binding of glutamate to the tRN A, performed with highly purified enzyme preparations, showed a K(m) v alue of 2.3 muM+/-0.3 for glutamate.