ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase

• Published in: Journal of biological inorganic chemistry Vol. 19; no. 8; pp. 1277 - 1285
• Main Authors: Nunes, Catarina I. P., Brás, Joana L. A., Najmudin, Shabir, Moura, José J. G., Moura, Isabel, Carepo, Marta S. P.
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-12-2014
• Subjects: Amino Acid Sequence; Arsenate Reductases - genetics; Arsenate Reductases - isolation & purification; Arsenate Reductases - metabolism; Bacillus subtilis; Biocatalysis; Biochemistry; Biomedical and Life Sciences; Calorimetry; Desulfovibrio; Desulfovibrio - enzymology; Escherichia coli; Kinetics; Life Sciences; Microbiology; Original Paper; Sequence Alignment; Staphylococcus aureus; Potassium-binding site; Sulfate-reducing bacteria; Kinetics; Thioredoxin; Arsenate reductase
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-014-1184-8

Desulfovibrio alaskensis
G20, a sulfate-reducing bacterium, contains an
arsRBC2C3
operon that encodes two putative arsenate reductases, DaG20_ArsC2 and DaG20_ArsC3. In this study, resistance assays in
E. coli
transformed with plasmids containing either of the two recombinant arsenate reductases, showed that only DaG20_ArsC3 is functional and able to confer arsenate resistance. Kinetic studies revealed that this enzyme uses thioredoxin as electron donor and therefore belongs to
Staphylococcus aureus
plasmid pI258 and
Bacillus subtilis
thioredoxin-coupled arsenate reductases family. Both enzymes from this family contain a potassium-binding site, but only in Sa_ArsC does potassium actually binds resulting in a lower
K
m
. Important differences between the
S. aureus
and
B. subtilis
enzymes and DaG20_ArsC3 are observed. DaG20_ArsC3 contains only two (Asn10, Ser33) of the four (Asn10, Ser33, Thr63, Asp65) conserved amino acid residues that form the potassium-binding site and the kinetics is not significantly affected by the presence of either potassium or sulfate ions. Isothermal titration calorimetry measurements confirmed nonspecific binding of K
+
and Na
+
, corroborating the non-relevance of these cations for catalysis. Furthermore, the low
K
m
and high
k
cat
values determined for DaG20_ArsC3 revealed that this enzyme is the most catalytically efficient potassium-independent arsenate reductase described so far and, for the first time indicates that potassium binding is not essential to have low
K
m
, for Trx-arsenate reductases.