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

Abstract

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology 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) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. 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 were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.

FIG. 1

Organization of sulfate activation genes in R. meliloti and E. coli. Regions of homology are blocked off with dotted lines. Domains that make up enzyme activities are bracketed and named at the bottom of the figure.

FIG. 2

Transcription start site of cysH_Rm. Primer extension experiments reveal two transcription start sites, SS1 and SS2. (a) Sequencing ladder (lanes 1 to 4), with primer extension reactions using Rm1021/pMW205 RNA grown in M9 minimal medium plus cysteine and methionine (lane 5) or M9 minimal medium alone (lane 6). (b) Region upstream of the ATG for cysH_Rm. The solid arrows represent SS1 and SS2, while the putative CysB binding site, the putative ribosome binding site (RBS), and the −10 and −35 regions are boxed and labeled. SS2 is upstream of the putative CysB binding site. (c) Structure of cysHDN_Rm. The small solid rectangle represents the putative CysB binding site.

FIG. 3

Alignment of the deduced amino acid sequences of the R. meliloti cysH gene product, the first 345 residues of the A. thaliana prh43 gene product, and the E. coli cysH gene product. Identical residues are boxed, and gaps are indicated by dashes. Sequences were aligned by the Clustal method in the program Lasergene.

FIG. 4

Sulfate activation from the R. meliloti cysteine regulon. (a) TLC plate analysis of several R. meliloti extracts for production of APS and PAPS. Lane 1 is a control reaction with no protein extract. The protein extracts were isolated from Rm1021 in M9 minimal medium (lane 2), JSS27 in M9 medium plus cysteine and methionine (lane 3), JSS27 in M9 medium plus cysteine (lane 4), JSS27 in M9 medium plus methionine (lane 5), JSS27 in M9 medium plus glutathione (lane 6), and MW26 in M9 medium plus methionine (lane 7). (b) Quantitation of the data from two experiments as in panel a.

FIG. 5

(a) Coomassie-stained gel of the His₆-CysH_Rm protein purification. Lanes 1 and 2 are elution fractions 1 and 2. The arrow indicates the His₆-CysH_Rm protein. (b) Radioactive TLC plate imaged with a phosphoimager, showing the preferential reduction of APS versus PAPS by the His₆-CysH_Rm protein. Either APS (lanes 1 to 7) or PAPS (lanes 8 to 14) was used as the substrate for His₆-CysH_Rm for 0, 1, 5, 10, 30, 60, or 120 min, respectively.

FIG. 6

Inhibition of His₆-CysH_Rm (0.312 mg/ml)-mediated reduction of 83 nM [³⁵S]APS by 5′-AMP (○) but not by 3′-AMP (✻). The standard reaction mixture was incubated for 1 h at 30°C, heat killed at 95°C for 3 min, and analyzed on a polyethyleneimine-cellulose TLC plate developed in 1 M LiCl. The intensity of each spot was quantitated with a phosphoimager. The percent sulfite was calculated as [(amount of sulfite signal)/(amount of sulfite signal + APS signal)] × 100.

FIG. 7

Requirement for thioredoxin but not glutathione in the His-CysH-mediated reduction of APS.