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J. Biol. Chem., Vol. 280, Issue 12, 11829-11839, March 25, 2005

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Glutathione Synthesis in Streptococcus agalactiae

ONE PROTEIN ACCOUNTS FOR -GLUTAMYLCYSTEINE SYNTHETASE AND GLUTATHIONE SYNTHETASE ACTIVITIES^*

Blythe E. Janowiak and Owen W. Griffith

From the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

-Glutamylcysteine synthetase (-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the -GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae -GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L--glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L--glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as -GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of -GCS-GS (total M_r 85,000) show 32% identity and 43% similarity with E. coli -GCS (M_r 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of -GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.

Received for publication, December 20, 2004 , and in revised form, January 3, 2005.

^* The studies reported were supported in part by an American Heart Association Predoctoral Fellowship Award (to B. E. J). A preliminary account of these studies was presented in March 2004 at the Oxygen Club of California World Congress in Santa Barbara, CA.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226. Tel.: 414-456-8778; Fax: 414-456-6510; E-mail: griffith{at}mcw.edu.

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