Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5′-adenylylsulfate reductase

Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5′-adenylylsulfate reductase

^*Biotech Center and Plant Science Department, Rutgers University, New Brunswick, NJ 08901-8250; and ^†Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115

Edited by Bob B. Buchanan, University of California, Berkeley, CA, and approved May 14, 1998 (received for review November 21, 1997)

Abstract

5′-Adenylylsulfate (APS) reductase (EC 1.8.99.-) catalyzes the reduction of activated sulfate to sulfite in plants. The evidence presented here shows that a domain of the enzyme is a glutathione (GSH)-dependent reductase that functions similarly to the redox cofactor glutaredoxin. The APR1 cDNA encoding APS reductase from Arabidopsis thaliana is able to complement the cysteine auxotrophy of an Escherichia coli cysH [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase] mutant, only if the E. coli strain produces glutathione. The purified recombinant enzyme (APR1p) can use GSH efficiently as a hydrogen donor in vitro, showing a K_m[GSH] of ≈0.6 mM. Gene dissection was used to express separately the regions of APR1p from amino acids 73–327 (the R domain), homologous with microbial PAPS reductase, and from amino acids 328–465 (the C domain), homologous with thioredoxin. The R and C domains alone are inactive in APS reduction, but the activity is partially restored by mixing the two domains. The C domain shows a number of activities that are typical of E. coli glutaredoxin rather than thioredoxin. Both the C domain and APR1p are highly active in GSH-dependent reduction of hydroxyethyldisulfide, cystine, and dehydroascorbate, showing a K_m[GSH] in these assays of ≈1 mM. The R domain does not show these activities. The C domain is active in GSH-dependent reduction of insulin disulfides and ribonucleotide reductase, whereas APR1p and R domain are inactive. The C domain can substitute for glutaredoxin in vivo as demonstrated by complementation of an E. coli mutant, underscoring the functional similarity between the two enzymes.

Footnotes

↵ ‡ To whom reprint requests should be addressed at: Biotech Center, 59 Dudley Road, Rutgers University, New Brunswick, NJ 08901-8250. e-mail: leustek{at}aesop.rutgers.edu.
This paper was submitted directly (Track II) to the Proceedings Office.
Abbreviations: GSH, reduced glutathione (when the reduced form is not specifically being referred to, the term “glutathione” is used); γ-EC, γ-glutamylcysteine; APS, 5′-adenylylsulfate; PAPS, 3′-phosphoadenosine-5′-phosphosulfate; DHA, dehydroascorbate; HED, hydroxyethyldisulfide.