Novel channel enzyme fusion proteins confer arsenate resistance

Abstract

Steady exposure to environmental arsenic has led to the evolution of vital cellular detoxification mechanisms. Under aerobic conditions, a two-step process appears most common among microorganisms involving reduction of predominant, oxidized arsenate (H(2)As(V)O(4)(-)/HAs(V)O(4)(2-)) to arsenite (As(III)(OH)(3)) by a cytosolic enzyme (ArsC; Escherichia coli type arsenate reductase) and subsequent extrusion via ArsB (E. coli type arsenite transporter)/ACR3 (yeast type arsenite transporter). Here, we describe novel fusion proteins consisting of an aquaglyceroporin-derived arsenite channel with a C-terminal arsenate reductase domain of phosphotyrosine-phosphatase origin, providing transposable, single gene-encoded arsenate resistance. The fusion occurred in actinobacteria from soil, Frankia alni, and marine environments, Salinispora tropica; Mycobacterium tuberculosis encodes an analogous ACR3-ArsC fusion. Mutations rendered the aquaglyceroporin channel more polar resulting in lower glycerol permeability and enhanced arsenite selectivity. The arsenate reductase domain couples to thioredoxin and can complement arsenate-sensitive yeast strains. A second isoform with a nonfunctional channel may use the mycothiol/mycoredoxin cofactor pool. These channel enzymes constitute prototypes of a novel concept in metabolism in which a substrate is generated and compartmentalized by the same molecule. Immediate diffusion maintains the dynamic equilibrium and prevents toxic accumulation of metabolites in an energy-saving fashion.

FIGURE 1.

A channel-enzyme fusion protein, Strop634, confers arsenate resistance in S. tropica. A, topology prediction of Strop634. Labeled are the Asn-Pro-Ala (NPA) signature motifs (filled blue circles), putative residues of the channel interior (red letters), the ArsC domain start (position 283), and the catalytic cysteines (filled red circles). The plot was generated using TeXtopo (22). B, structure model of Strop634 and Strop1447 depicting the size relation of the channel and ArsC domains based on the E. coli aquaglyceroporin (Protein Data Bank code 1FX8) and ArsC (Protein Data Bank code 1J9B). C, domain arrangement of Strop634 and Strop1447, the latter carrying a single cysteine. The relative positions of the cysteines (C) are labeled above the bars. D, Western blot showing full-length expression of recombinant Strop634 in yeast (rStrop634), as well as Strop634 and Strop1447 in S. tropica using specific antisera directed against either reductase domain. E, arsenate sensitivity of S. tropica WT and deletion strains. 10-fold serial dilutions of S. tropica spores were spotted on agar medium containing 0, 0.1, or 0.5 mm arsenate and were incubated at 29 °C for 5 days.

FIGURE 2.

Functional assessment of Strop634, Strop1447, and Rv2643 in yeast deletion strains. A, complementation of Δacr2 yeast by expression of Strop634, Strop1447, and their separate ArsC domains (dom.). Cells were spotted on 0, 1, and 2 mm arsenate media and incubated at 29 °C for 4 days. B, assay for inward arsenite permeability. Expression of functional arsenite channels in Δacr2,3 Δfps1 yeast leads to reduced growth on 1 and 2 mm arsenite media. C, complementation of a highly arsenate sensitive yeast strain (Δacr2,3 Δfps1 Δycf1). Yeast growth indicates both reduction of arsenate and outward arsenite permeability. Cells expressing Strop634, Strop1447, Rv2643, and Strop chimeras with swapped channel and reductase domains were spotted on 0, 20, and 40 μm arsenate media.

FIGURE 3.

Direct arsenite, water, and glycerol permeability assays. A, shown is the accumulation of arsenic in 1 mm arsenite-exposed Δacr2,3 Δfps1 Δycf1 yeast cells expressing Fps1 (+ control; orange triangles), Strop634 (light blue circles), Strop1447 (dark blue circles), or in nonexpressing cells (− control; black triangles). The error bars denote S.E. (n = 3). B, dynamic light scattering in a hyperosmotic, inward directed 300 mm glycerol gradient of Δfps1 yeast protoplasts expressing PfAQP (+ control; orange trace), Strop634 (light blue trace), Strop1447 (dark blue trace), or of nonexpressing protoplasts (− control; black trace). The first phase indicates osmotic water release, and the second phase indicates glycerol uptake. 10 traces were averaged per experiment. The insert shows water (P_f; open bars; left ordinate) and glycerol permeability coefficients (P_gly; filled bars; right ordinate) calculated from the traces. The error bars denote S.E. (n = 10). Asterisks label values that are significant (p < 0.5) compared with the − control.

FIGURE 4.

In vitro analysis of Strop634 and Strop1447 ArsC activity. The Michaelis-Menten plot shows the arsenate-dependent rate of NADPH oxidation by a mixture of Strop634 (filled circles) or Strop1447 (open circle) with thioredoxin and thioredoxin reductase. The insert depicts the rate of 4-nitrophenyl phosphate hydrolysis catalyzed by Strop634 (filled circle) and Strop1447 (open circle).

FIGURE 5.

Evidence for Strop1447 function in S. tropica arsenate resistance and models of channel enzyme functionality. A, S. tropica WT as well as strains with full deletion of strop634 or a partial deletion of the strop634 ArsC domain (dom.) were spotted on arsenate media. The strop1447 gene was left intact. B, derived models of dual-functional proteins in arsenate resistance using thioredoxin (Trx) or putatively mycoredoxin/mycothiol (Mrx/MySH).