Corynebacterium glutamicum survives arsenic stress with arsenate reductases coupled to two distinct redox mechanisms

Abstract

SummaryArsenate reductases (ArsCs) evolved independently as a defence mechanism against toxic arsenate. In the genome of Corynebacterium glutamicum, there are two arsenic resistance operons (ars1 and ars2) and four potential genes coding for arsenate reductases (Cg_ArsC1, Cg_ArsC2, Cg_ArsC1' and Cg_ArsC4). Using knockout mutants, in vitro reconstitution of redox pathways, arsenic measurements and enzyme kinetics, we show that a single organism has two different classes of arsenate reductases. Cg_ArsC1 and Cg_ArsC2 are single-cysteine monomeric enzymes coupled to the mycothiol/mycoredoxin redox pathway using a mycothiol transferase mechanism. In contrast, Cg_ArsC1' is a three-cysteine containing homodimer that uses a reduction mechanism linked to the thioredoxin pathway with a k cat/KM value which is 10 3 times higher than the one of Cg_ArsC1 or Cg_ArsC2. Cg_ArsC1' is constitutively expressed at low levels using its own promoter site. It reduces arsenate to arsenite that can then induce the expression of Cg_ArsC1 and Cg_ArsC2. We also solved the X-ray structures of Cg_ArsC1' and Cg_ArsC2. Both enzymes have a typical low-molecular-weight protein tyrosine phosphatases-I fold with a conserved oxyanion binding site. Moreover, Cg_ArsC1' is unique in bearing an N-terminal three-helical bundle that interacts with the active site of the other chain in the dimeric interface.

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

“…Mrx1 reacts with the latter, releasing As(III) and forming a mixed disulfide with MSH which is then reduced by a second molecule of MSH yielding reduced Mrx1 and mycothiol disulfide. For MtAhpE-SS-M reduction by MtMrx1, we propose an analogous monothiolic mechanism (16,33), as illustrated in Fig. 7 described below.…”

“…MtMrx1-In C. glutamicum Mrx1, only the N-terminal cysteine residue of the CXXC active site sequence motif was found to be essential for the reduction of the arsenate-mycothiol adduct intermediate (16,33). For the MtMrx1, however, the C-terminal cysteine mutated to alanine (MtMrx1 CXXA) was not able to reduce MtAhpE.…”

Protein S-Mycothiolation Functions as Redox-Switch and Thiol Protection Mechanism in Corynebacterium glutamicum Under Hypochlorite Stress

“…After different times of stress exposure, the glucose uptake reaction was then started by addition 50 lM [ 14 C]-glucose (specific activity 250 lCi lmol -1 ; Moravek Biochemicals). At given time intervals (15,30,45,60, and 90 s), 200 ll samples were filtered through glass fibre filters (Typ F; Millipore) and washed twice with 2.5 ml of 100 mM LiCl. Radioactivity of the samples was determined using scintillation fluid (Rotiszinth; Roth) and a scintillation counter (LS 6500; Beckmann).…”

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

“…Arsenate reduction to arsenite is catalyzed by arsenate reductase, an enzymatic activity carried out by at least three non-related families. These reductases use the thioredoxin, glutaredoxin or mycoredoxin systems as electron donors [20], [21], [22], [23], [24], [25], [26]. Arsenite export is mediated by two families of proteins: ArsB proteins, that are present only in bacteria [27], and Acr3 proteins, which are more widely distributed in different organisms including bacteria, fungi and plants [28], [29]).…”