| 2.A.59 The Arsenical Resistance-3 (ACR3) Family
Two proteins of the ACR3 family have been functionally characterized. These proteins are the ACR3 protein of Saccharomyces cerevisiae, also called the ARR3 protein (Wysocki et al., 1997), and the 'ArsB' protein of Bacillus subtilis (Sato and Kobayashi, 1998). The latter protein is not related to ArsB of E. coli. The former yeast protein is present in the plasma membrane and pumps arsenite and antimonite, but not arsenate, tellurite, cadmium or phenylarsine oxide out of the cell in response to the pmf. It uses a proton antiport mechanism to extrude the anions with low affinity (Maciaszczyk-Dziubinska et al., 2011). The Bacillus protein exports both arsenite and antimonite. The exact transport mechanism is not established. Bacillus Acr3 probably has a 10 TMS topology (Aaltonen and Silow, 2008).
ACR3 of S. cerevisiae is 404 amino acyl residues long and exhibits 10 putative(TMSs. Homologues are found in Mycobacterium tuberculosis (498 aas; gbZ80225), Archaeoglobus fulgidus (370 aas; gbAE001071), Methanobacterium thermoautotrophicum (365 aas; gbAE000865) and Synechocystis (383 aas; spP74311). Thus, members of the ACR3 family are found in bacteria, archaea and eukarya. Sequence similarity of several members of the Acr3 family with members of the bile acid:Na+ symporter (BASS) family (TC #2.A.28) is sufficient to establish homology. The ACR3 family belongs to the BART superfamily (Mansour et al., 2007). When an ACR3 protein functions with an ATPase, that ATPase is a member of the ArsA superfamily.
Bioinformatic analyses have suggested that some ACR3 porters are encoded within operons together with ArsA-like ATPases, implying that some of these porters may be driven by ATP hydrolysis as primary active transporters (Castillo and Saier, 2010). This may occur in addition to or instead of the secondary active transport mechanism established for ACR3 members noted above. Homologous ATPases are found in families 3.A.4, 3.A.19 and 3.A.21 as well as 2.A.59. A region of the ABC ATPase (3.A.1.26.8; the ribose transporter) shows significant sequence similarity to the ArsA under 3.A.19.1.1 (28% identical; 49% similarity, 0 gaps, e-5) as revealed by TC Blast.
ACR3 family members confer high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3(Bs) ) exhibits a typical NhaA structural fold, with two discontinuous helices of TMSs 4 and 9 interacting with each other and forming an X-shaped structure. Lv et al. 2022 investigated the evolutionary conservation among 300 homologous sequences and identified three conserved motifs in both the discontinuous helices and TMS5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TMS9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TMS4 and E322 in TMS10 are responsible for structural stabilization. The highly conserved residues on Motif B of TMS5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporter (Lv et al. 2022).
The generalized reaction catalyzed by members of the ACR3 family is:
Arsenite or antimonite (in)  Arsenite or antimonite (out)
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This family belongs to the IT Superfamily.
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| References: |
Aaltonen, E.K., and M. Silow. (2008). Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis. Biochim. Biophys. Acta. 1778: 963-973.
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Baker-Austin, C., M. Dopson, M. Wexler, R.G. Sawers, A. Stemmler, B.P. Rosen, and P.L. Bond. (2007). Extreme arsenic resistance by the acidophilic archaeon 'Ferroplasma acidarmanus' Fer1. Extremophiles 11: 425-434.
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Castillo, R. and M.H. Saier. (2010). Functional Promiscuity of Homologues of the Bacterial ArsA ATPases. Int J Microbiol 2010: 187373.
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Fu, H.L., B.P. Rosen, and H. Bhattacharjee. (2010). Biochemical characterization of a novel ArsA ATPase complex from Alkaliphilus metalliredigens QYMF. FEBS Lett. 584: 3089-3094.
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Fu, H.L., Y. Meng, E. Ordóñez, A.F. Villadangos, H. Bhattacharjee, J.A. Gil, L.M. Mateos, and B.P. Rosen. (2009). Properties of arsenite efflux permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum. J. Biol. Chem. 284: 19887-19895.
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Lv, P., Y. Shang, Y. Zhang, W. Wang, Y. Liu, D. Su, W. Wang, C. Li, C. Ma, and C. Yang. (2022). Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern. FASEB J. 36: e22659.
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Maciaszczyk-Dziubinska E., Migocka M., Wawrzycka D., Markowska K. and Wysocki R. (2014). Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae. Biochim Biophys Acta. 1838(3):747-55.
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Maciaszczyk-Dziubinska, E., M. Migocka, and R. Wysocki. (2011). Acr3p is a plasma membrane antiporter that catalyzes As(III)/H+ and Sb(III)/H+ exchange in Saccharomyces cerevisiae. Biochim. Biophys. Acta. 1808: 1855-1859.
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Mansour, N.M., M. Sawhney, D.G. Tamang, C. Vogl, and M.H. Saier, Jr. (2007). The bile/arsenite/riboflavin transporter (BART) superfamily. FEBS J. 274: 612-629.
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Markowska K., Maciaszczyk-Dziubinska E., Migocka M., Wawrzycka D. and Wysocki R. (2015). Identification of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae As(III)/H+ antiporter. Mol Microbiol. 98(1):162-74.
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Ramos, J., J. Ariño, and H. Sychrová. (2011). Alkali-metal-cation influx and efflux systems in nonconventional yeast species. FEMS Microbiol. Lett. 317: 1-8.
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Sato, T. and Y. Kobayashi. (1998). The ars operon in the skin element of Bacillus subtilis confers resistance to arsenate and arsenite. J. Bacteriol. 180: 1655-1661.
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Villadangos, A.F., H.L. Fu, J.A. Gil, J. Messens, B.P. Rosen, and L.M. Mateos. (2012). Efflux permease CgAcr3-1 of Corynebacterium glutamicum is an arsenite-specific antiporter. J. Biol. Chem. 287: 723-735.
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Wawrzycka, D., K. Markowska, E. Maciaszczyk-Dziubinska, M. Migocka, and R. Wysocki. (2016). Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae. Biochim. Biophys. Acta. 1859: 117-125. [Epub: Ahead of Print]
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Wysocki, R., P. Bobrowicz, and S. Ulaszewski. (1997). The Saccharomyces cerevisiae ACR3 gene encodes a putative membrane protein involved in arsenite transport. J. Biol. Chem. 272: 30061-30066.
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Xia, X., V.L. Postis, M. Rahman, G.S. Wright, P.C. Roach, S.E. Deacon, J.C. Ingram, P.J. Henderson, J.B. Findlay, S.E. Phillips, M.J. McPherson, and S.A. Baldwin. (2008). Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter. Mol. Membr. Biol. 25: 691-705.
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Yang, H.C., H.L. Fu, Y.F. Lin, and B.P. Rosen. (2012). Pathways of arsenic uptake and efflux. Curr Top Membr 69: 325-358.
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 2.A.59.1.1 | Plasma membrane ACR3 arsenite exporter (AsIII:H+ antiporter) (Wysocki et al., 1997). Catalyzes proton antiport with either arsenite or antimonite. Cys151 is essential for transport activity, while Cys90 and Cys169 are important in trafficking (Maciaszczyk-Dziubinska et al. 2013). Other residues important for folding, trafficking and/or activity have also been identified (Markowska et al. 2015). This protein has 10 TMSs with cytoplasmically oriented N- and C-termini, and a homology-based structural model of Acr3 using the
crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter,
ASBT, has been constructed (Wawrzycka et al. 2016). | Fungi | ACR3 of Saccharomyces cerevisiae |
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| 2.A.59.1.2 | ArsB arsenite/antimonite exporter (Sato and Kobayashi, 1998) | Yeast, bacteria, archaea | ArsB of Bacillus subtilis (spP45946) |
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| 2.A.59.1.3 | The arsenical resistance membrane protein, the arsenate (not arsenite) transporter, So_ACR3 (Xia et al., 2008) (with 7 putative TMSs and an N-terminus in the periplasm).
| Bacteria | ACR3 of Shewanella oneidensis (Q8EJD3) |
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| 2.A.59.1.4 | Arc3 homologue | Archaea | Acr3 of Pyrococcus furiosus (Q8U3B8) |
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| 2.A.59.1.5 | The arsenite-specific exporter, Acr3/ArsA1/ArsA2. ArsA1 and ArsA2 are half sized ATPases (Fu et al., 2010). | Bacteria | The Acr3/ArsA1/ArsA2 arsenite exporter of Alkaliphilus metalliredigens
Acr3 (A6TP80)
ArsA1 (A6TP82)
ArsA2 (A6TP83) |
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| 2.A.59.1.6 | The arsenite-specific exporter, Acr3'/ArsA1'/ArsA2'. ArsA1' and ArsA2' are half sized ATPases (Fu et al., 2009; 2010) | Bacteria | The Acr3'/ArsA1'/ArsA2' arsenite exporter of Alkaliphilus metalliredigens
Acr3' (A6TLY3)
ArsA1' (A6TLY5)
ArsA2' (A6TLY6) |
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| 2.A.59.1.7 | Aresenite (AsIII):H+ antiporter, Acr3-1; confers resistance to As(III) but not Sb(III) (antimonite) (Villadangos et al. 2012; Yang et al. 2012).
| Actinobacteria | Acr3-1 of Corynebacterium glutamicum |
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| Examples: |
| TC# | Name | Organismal Type | Example |
| 2.A.59.2.1 | Putative efflux pump (Acr3) | Bacteria | Acr3 of Sulfitobacter sp. NAS-14.1 (A3T1V5)
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| 2.A.59.2.2 | Acr3 homologue of unknown function (10 putative TMSs) | Bacteria | Acr3 of Chloroflexus aggregans (B8GAD2)
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| Examples: |
| TC# | Name | Organismal Type | Example |