Arsenate reductase of Staphylococcus aureus plasmid pI258.
Arsenate reductase encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 was overproduced in Escherichia coli and purified. The purified enzyme reduced radioactive arsenate to arsenite when coupled to thioredoxin, thioredoxin reductase, and NADPH. NADPH oxidation coupled to arsenate reduction also required thioredoxin and thioredoxin reductase. Glutaredoxin and reduced glutathione did not stimulate arsenate reduction. NADPH oxidation showed Michaelis-Menten kinetics with a Km of 1 microM AsO4(3-) and an apparent Vmax of 200 nmol/min per mg of protein. At high substrate concentration (above 1 mM AsO4(3-), a secondary rise in the reaction rate was observed, with a Km of 2 mM and an apparent Vmax of 450 nmol/min per mg of protein. This secondary rise also occurred upon addition of phosphate or nitrate (which were not substrates for the enzyme). Arsenite (the product of the enzyme), tellurite, and antimonite [Sb(III)] were inhibitors. Selenate (but not selenite or sulfate) was a substrate for reductase-dependent NADPH oxidation, with an apparent Km of 13 mM SeO4(2-). Arsenate reductase was purified as a monomer of 14.5 kDa, consistent with the DNA sequence. Electrospray mass spectrometry showed two molecular masses of 14,810.5 and 14,436.0 Da, suggesting that 70% of the purified protein lacked the N-terminal three amino acids; HPLC coupled to electrospray mass spectroscopy of protease digest products confirmed this conclusion and verified the entire amino acid sequence.
Ji
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Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612-7344.
Garber
E A
EA
Armes
L G
LG
Chen
C M
CM
Fuchs
J A
JA
Silver
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S
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D053501:Arsenite Transporting ATPases; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D016623:Ion Pumps; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017433:Protein Structure, Secondary; D064586:Selenic Acid; D018036:Selenium Compounds / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D016623:Ion Pumps; D009097:Multienzyme Complexes; D009838:Oligodeoxyribonucleotides; D018036:Selenium Compounds; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; D064586:Selenic Acid