A New Strategy for As(V) Biosensing Based on the Inhibition of the Phosphatase Activity of the Arsenate Reductase from Thermus thermophilus

Abstract

Arsenic (As) pollution is a widespread problem worldwide. In recent years, biosensors based on enzymatic inhibition have been developed for arsenic detection, making the study of the effect of inhibitors on the selected enzymatic activity crucial for their setup. The arsenate reductase of Thermus thermophilus HB27, TtArsC, reduces As(V) into As(III), but is also endowed with phosphatase activity. This work investigates the inhibitory effects of As(V) and As(III) on phosphatase activity by taking advantage of a simple colorimetric assay; the results show that both of them are non-competitive inhibitors affecting the Vmax but not the KM of the reaction. However, their Ki values are different from each other (15.2 ± 1.6 μm for As(V) and 394.4 ± 40.3 µm with As(III)), indicating a higher inhibitory effect by As(V). Moreover, the inhibition-based biosystem results to be selective for As(V) since several other metal ions and salts do not affect TtArsC phosphatase activity; it exhibits a sensitivity of 0.53 ± 0.03 mU/mg/μm and a limit of detection (LOD) of 0.28 ± 0.02 μm. The good sensitivity and specificity for As(V) point to consider inhibition of TtArsC phosphatase activity for the setup of a novel biosensor for the detection of As(V).

Structural and functional mapping of ars gene cluster in Deinococcus indicus DR1

“…This family is related to the low molecular weight protein-tyrosine phosphatases (LMW PTPases) family protein [70] . Arsenate reductase of B. subtilis, S. aureus plasmid pI258 and T. thermophilus HB27 can also display weak phosphatase activity [66] , [70] , [85] . Recently, it was shown in T. thermophilus HB27 that both arsenate and arsenite can inhibit the phosphatase activity through non-competitive mode of inhibition [85] .…”

“…Arsenate reductase of B. subtilis, S. aureus plasmid pI258 and T. thermophilus HB27 can also display weak phosphatase activity [66] , [70] , [85] . Recently, it was shown in T. thermophilus HB27 that both arsenate and arsenite can inhibit the phosphatase activity through non-competitive mode of inhibition [85] . Our results indicate that both the arsenate reductases of ars gene cluster i.e., ArsC2 and ArsC3 of D. indicus belong to the LMW PTPases family protein and probably utilize thioredoxin.…”

Extremozyme-Based Biosensors for Environmental Pollution Monitoring: Recent Developments

“…[ 82 , 97 , 103 , 195 , 196 , 197 ]. The catalytic activity of arsenite oxidase [ 198 ] and arsenate reductase [ 134 ] served, in addition, for the specific detection of arsenic species. Many biosensors are characterized by detection limits in the nanomolar range and a few of them even enable the detection of heavy metals in drinking water at ppb levels, compatible with the current MRLs [ 82 ].…”

“…Enzyme-based biosensors based on mesophilic enzymes were developed based on the principle of enzymatic inhibition of, e.g., acetylcholinesterase and phosphatase [ 199 , 200 ], or based on the direct detection, e.g., the detection of arsenite using arsenite oxidase biosensors [ 198 ]. More recently, the detection of arsenic was addressed with an arsenate reductase extracted from Thermus thermophilus HB27 (TtArsC) [ 132 , 133 , 134 ] and with two thermostable protein chimeras [ 133 ].…”