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

被引:100
作者
Bick, JA
Åslund, F
Chen, YC
Leustek, T
机构
[1] Rutgers State Univ, Biotech Ctr, New Brunswick, NJ 08901 USA
[2] Rutgers State Univ, Dept Plant Sci, New Brunswick, NJ 08901 USA
[3] Harvard Univ, Sch Med, Dept Microbiol & Mol Genet, Boston, MA 02115 USA
关键词
D O I
10.1073/pnas.95.14.8404
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 approximate to 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 approximate to 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,
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页码:8404 / 8409
页数:6
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