Reaction Mechanism of Thioredoxin: 3′‐Phospho‐adenylylsulfate Reductase Investigated by Site‐Directed Mutagenesis

Abstract

Properties of purified recombinant adenosine 3'-phosphate 5'-phosphosulfate (PAdoPS) reductase from Escherichia coli were investigated. The Michaelis constants for reduced thioredoxin and PAdoPS are 23 pM and 10 pM, respectively; the enzyme has a V,,,,,, of 94-99 pmol min.. mg and a molecular activity/catalytically active dimer of 95 s-'. Adenosine 3',5'-bisphosphate (PAdoP) inhibits competetively ( K , 4 pM) with respect to PAdoPS; adenosine 2',5'-bisphosphate and sulfite are not inhibitory. Alkylation by SH-group inhibitors irreversibly inactivates the enzyme.The structural gene (cysH) encodes for a small polypeptide with a single Cys residue located in a conserved cluster (KXECGVLH) of amino acids. Involvement of the only Cys and of Tyr209 in the reduction of PAdoPS to sulfite was investigated by site-specific mutagenesis: cysH was mutated by single-strand-overlay extension PCR; the mutated genes were cloned in pBTacl and expressed in E. coli RL 22 (AcysHIJ). Homogenous Cys239Ser and Tyr209Phe mutant PAdoPS reductases were investigated for altered catalytic properties. Mutation of the single Cys reduced V,,,,, by a factor of 4.5X 10' (V,,.,, = 0.02-0.013 pmol min-' mg ') with marginal effects on K,, for PAdoPS (19 pM) and reduced thioredoxin (14 pM). Mutation of Tyr209 drastically affected saturation with thioredoxin (K,,, 1 .S pM) and decreased V,,,.,, (0.22-0.25 pmol min-' mg I ) in addition to a small increase in K,, for PAdoPS (31 pM).Chromophores as prosthetic groups were absent from recombinant PAdoPS reductase. Difference absorption spectra between reduced and oxidized forms of wild-type and mutated proteins indicated that, in addition to Cys239 and Tyr209, an unidentified Trp (Ain,:,x 292 nm) appears to be involved in the reduction.The data suggest a special ping-pong mechanism with PAdoPS reacting with the reduced enzyme isomer in a Theorell-Chance type mechanism.

A yeast two-hybrid knockout strain to explore thioredoxin-interacting proteinsin vivo

“…This result first confirmed that C245 is the essential cysteine for MET16 interaction with TRXs (47). Second, this experiment demonstrated that the CY306 strain is a functional and reliable tool to discriminate cysteines targeted by TRXs in vivo.…”

“…We finally investigated the ability of the CY306 strain to reveal critical residues such as cysteines involved in TRX interaction with its target. We took as an example the MET16 enzyme that contains two cysteine residues: C112, which does not possess any catalytic activity (46), and C245 in the consensus sequence KxECG(L͞I)H, which is required for MET16 activity (47). Before this work, the identity of the cysteine residue targeted by TRX in MET16 was unknown.…”

The Presence of an Iron-Sulfur Cluster in Adenosine 5′-Phosphosulfate Reductase Separates Organisms Utilizing Adenosine 5′-Phosphosulfate and Phosphoadenosine 5′-Phosphosulfate for Sulfate Assimilation

“…All of these proteins are characterized by a highly conserved (KRT)ECG(LI)H motif ( Fig. 1) containing a catalytically active Cys residue (32,33). In addition, a number of related proteins of unknown function were found in several archaebacteria, including Methanococcus jannaschii, Pyrococcus horikoshii, and Pyrococcus abyssii.…”

Sulfate assimilation pathway intermediate phosphoadenosine 5′-phosphosulfate acts as a signal molecule affecting production of curli fibres in Escherichia coli

“…As expected, all mutants were auxotrophic for cysteine (Table 2); however, they displayed WT phenotypes on Congo red and calcofluor plates, with the partial exception of the cysQ mutant, showing some fluorescence on calcofluor medium (Fig. 2a) carrying either a WT cysH gene or a mutant allele, in which two codons encoding C-terminal amino acids identified as the redox-active centre of the enzyme (Berendt et al, 1995) had been substituted to obtain two alanine residues (pT7cysH wt and pT7cysH mut , respectively). Production of both the WT and mutated CysH proteins from the pT7-7 plasmid derivatives was clearly detectable on SDS-PAGE (Fig.…”

“…For expression of WT CysH protein, the corresponding gene was amplified by PCR from the E. coli MG1655 chromosome using primers cysH_NdeI_for and cysH_PstI_rev, and the resulting product was cloned into pT7-7 vector using the NdeI/ PstI restriction sites. The pT7cysH mut plasmid, carrying a mutant cysH allele encoding a protein with a non-functional redox-active centre (Berendt et al, 1995), was constructed by amplifying the cysH gene using primers cysH_NdeI_for and cysH_mut_PstI_rev, resulting in the following substitutions: TAG at nt 715, GAC at nt 716, CAG at nt 724, AAC at nt 725. The four mutations resulted in substitution of both Cys240 and His243 to alanine residues (ECGLHAEAGLA).…”