The conversion of -glutamate to -glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with ␣-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for ␣-glutamate than -glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards -glutamate were much smaller than rates with the ␣-isomer. These results are discussed in light of the observation that -glutamate is accumulated as an osmolyte in many archaea while -glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.The glutamate-to-glutamine conversion by glutamine synthetase (GS) provides the nitrogen donor for the first step in the biosynthesis of many amino acids, purines, pyrimidines, and amino sugars. In most bacteria and archaea GS is a multimeric enzyme consisting of 12 identical subunits, each with a mass of 50 to 55 kDa (12). Modes of regulation further classify the enzyme as GSI-␣ (not subject to adenylylation [4]) or GSI- activities (as exemplified by the Escherichia coli GS [12,23]). GS appears to have another unusual role in one particular archaeon. The halophilic methanogen Methanohalophilus portucalensis uses -glutamine as an osmolyte when grown in media containing Ͼ2 M NaCl (10,17,19). Synthesis of -glutamine in this organism has been suggested to occur via GS action on -glutamate (16). M. portucalensis is the only organism known to date to accumulate this zwitterionic -amino acid, although -glutamate is a substrate for sheep brain and rat liver GS (14). The ability of GS to use -glutamate as a substrate may be a characteristic of other archaea or of a wider range of bacteria, or it may be a property only associated with the halophilic methanogen. In order to examine this, GS from M. portucalensis was partially purified and its activity toward both ␣-and -glutamate was examined. The selectivity of ␣-over -glutamate is much lower for the M. portucalensis GS than for the GS from the other archaeal (cloned and purified enzymes from Methanococcus jannaschii and Archaeoglobus fulgidus) and bacterial (E. coli and Bacillus subtilis) enzyme activities examined. This enhanced relative activity toward -glutamate is consistent with an involvement of the enzyme in the accumulation of -glutamine in high-NaCl environments in M. portucalensis. These results are discussed in terms of the role of -glutamate and -glutamine as osmolytes in archaea.
MATERIALS AND METHODSChemicals. Tris-HCl, MgCl 2 , -mercaptoethanol, ␣-glutamate, -glutamate, and NH 4 Cl were obtained from Sigma. Q-Sepharose Fast Flow anion exchange resin was purchased from Pharmacia.Enzymes. The A. fulgidus VC-16 GS structural gene was ...