The Putative Moss 3′-Phosphoadenosine-5′-phosphosulfate Reductase Is a Novel Form of Adenosine-5′-phosphosulfate Reductase without an Iron-Sulfur Cluster

Abstract

Sulfate assimilation provides reduced sulfur for synthesis of the amino acids cysteine and methionine and for a range of other metabolites. Sulfate has to be activated prior to reduction by adenylation to adenosine 5-phosphosulfate (APS). In plants, algae, and many bacteria, this compound is reduced to sulfite by APS reductase (APR); in fungi and some cyanobacteria and ␥-proteobacteria, a second activation step, phosphorylation to 3-phosphoadenosine 5-phosphosulfate (PAPS), is necessary before reduction to sulfite by PAPS reductase (PAPR). We found previously that the moss Physcomitrella patens is unique among these organisms in possessing orthologs of both APR and PAPR genes (Koprivova, A., Meyer, A. J., Schween, G., Herschbach, C., Reski, R., and Kopriva, S. (2002) J. Biol. Chem. 277, 32195-32201). To assess the function of the two enzymes, we compared their biochemical properties by analysis of purified recombinant proteins. APR from Physcomitrella is very similar to the well characterized APRs from seed plants. On the other hand, we found that the putative PAPR preferentially reduces APS. Sequence analysis, analysis of UV-visible spectra, and determination of iron revealed that this new APR, named PpAPR-B, does not contain the FeS cluster, which was previously believed to determine the substrate specificity of the otherwise relatively similar enzymes. The lack of the FeS cluster in PpAPR-B catalysis is connected with a lower turnover rate but higher stability of the protein. These findings show that APS reduction without the FeS cluster is possible and that plant sulfate assimilation is predominantly dependent on reduction of APS.Sulfur is essential for life. It is present in the amino acids cysteine and methionine as part of proteins and peptides; in many coenzymes and prosthetic groups, such as FeS centers, thiamine, lipoic acid, etc.; and in a variety of secondary metabolites, e.g. glucosinolates and alliins in plants. In these compounds, sulfur is present in the reduced form of organic thiols; however, the major form of sulfur available in nature is sulfate.

Targeted knock‐out of a gene encoding sulfite reductase in the moss Physcomitrella patens affects gametophytic and sporophytic development

“…The same co-localization was obtained with a control construct encoding GFP fused to the chloroplast targeting peptide of FtsZ2-1 [28]. The fluorescence signal of the PpSiR isoforms was not uniformly distributed within the plastids but was concentrated in multiple spots, similar to the localization APS reductase [20]. All three SiR genes are expressed in protonema and gametophores as revealed by EST databases and RT-PCR (data not shown).…”

“…The intracellular localization of SiR isoforms was addressed by transient expression of vectors encoding C-terminal green fluorescent protein (GFP) fusion proteins as described in [20]. The complete open reading frames of PpSiR1, PpSiR2 and PpSiR3 were fused to GFP reporter using the plasmid mAV4 [27].…”

“…As a control for plastid targeting, the plasmid pCTP-GFP [28] was used. The transfection and microscopy was performed as in [20].…”

“…Analysis of sulfur metabolism in P. patens revealed that some metabolic steps are less complex in this moss than in seed plants, while others are more diverse in mosses [19]. For example, for the reduction of activated sulfate (adenosine 5 0 -phosphosulfate, APS) to sulfite, a novel form of APS reductase without an iron-sulfur cofactor was identified in P. patens [20,22]. Also, the reduction of sulfite to sulfide by sulfite reductase (SiR) is more complex in Physcomitrella than in seed plants.…”

“…patens has been widely used to address various questions of primary metabolism, such as fatty acid synthesis, N-glycosylation, or sulfur metabolism [10,11,[17][18][19][20]. Many such analyses revealed an unexpected variety of enzymes and pathways alternative to those in flowering plants.…”