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)