SACCHAROMYCES-CEREVISIAE HAS A SINGLE GLUTAMATE SYNTHASE GENE CODING FOR A PLANT-LIKE HIGH-MOLECULAR-WEIGHT POLYPEPTIDE

Purification of the glutamate synthase (GOGAT) enzyme from Saccharomyc es cerevisiae showed that it is an oligomeric enzyme composed of three identical 199-kDa subunits. The GOGAT structural gene was isolated by screening a yeast genomic library with a yeast PCR probe. This probe was obtained by amplification with degenerate oligonucleotides designe d from conserved regions of known GOGAT genes, The derived aminotermin al sequence of the GOGAT gene was confirmed by direct amino-terminal s equence analysis of the purified protein of 199 kDa. Northern (RNA) an alysis allowed the identification of an mRNA of about 7 or 8 kb. An in ternal fragment of the GOGAT gene was used to obtain null GOGAT mutant s completely devoid of GOGAT activity. The results show that S. cerevi siae has a single NADH-GOGAT enzyme, consisting of three 199-kDa monom ers, that differs from the one found in prokaryotic microorganisms but is similar to those found in other eukaryotic organisms such as alfal fa.