Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone

Abstract

The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface that affect their interactions.

Genome sequence of Pseudomonas aeruginosa PA1-Petro—A role model of environmental adaptation and a potential biotechnological tool

“…The ArsA ATPase, the catalytic subunit of the ArsAB As(III) efflux pump, receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations ( Vahter, 2002 ; Yang et al., 2011 ; Pillai et al., 2014 ). In this study, we also identified a putative transcriptional regulator in PA1-Petro only.…”

“…On the other hand, some researchers argue that the use of tellurium compounds may negatively affect the immune defense, interfering with the recruitment of neutrophils against bacteria ( Nguyen et al., 2013 ). Now, Arsenic enters the environment from both geochemical and anthropogenic sources, representing a global health issue affecting millions of people worldwide through environmental and occupational exposure ( Pillai et al., 2014 ; Kuivenhoven and Mason, 2021 ). It is a naturally occurring metalloid element, also found in the air and food products ( Carlin et al., 2016 ).…”

NemA Catalyzes Trivalent Organoarsenical Oxidation and Is Regulated by the Trivalent Organoarsenical-Selective Transcriptional Repressor NemR

“…The quenching of intrinsic protein fluorescence has been used to examine the binding specificity in proteins that bind to trivalent arsenicals, including ArsA ATPase, , ArsD As­(III)/Sb­(III) metallochaperone, and ArsM As­(III) S -adenosylmethionine methyltransferase . NemR has one tryptophan residue, Trp167 (Figure S5), which is responsible for the majority of its intrinsic fluorescence.…”

Metagenomic and genomic analysis of heavy metal-tolerant and -resistant bacteria in resource islands in a semi-arid zone of the Colombian Caribbean

“…The molecular function of the protein is associated with the active transmembrane transporter of arsenite (Table S2 ). The product of the arsA gene is an arsenite-stimulated ATPase that, when associated with arsB, forms a complex that functions as an anion-translocating ATPase (Pillai et al 2014 ). Additionally, higher taxonomic abundances were observed during the wet season (Fig.…”