Reduction of adenosine-5 '-phosphosulfate instead of 3 '-phosphoadenosine-5 '-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and othermembers of the family Rhizobiaceae

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizo bium meliloti) that are involved in sulfate activation for cysteine biosynt hesis. Two of the genes display homology to the Escherichia coil cysDN gene s, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homol ogy to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS ) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) r eductase. In order to determine the specificity of the R. meliloti reductas e, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the hist idine-tagged R. meliloti cysH gene product appears to favor APS over PAPS a s a substrate, with a K-m for APS of 3 to 4 mu M but a K-m for PAPS of >100 mu M. In order to determine whether this preference for APS is unique to R . meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens mere assayed for APS or PAPS reductase activity. Cell extracts from all four species als o preferentially reduce APS over PAPS.