Overexpression of a Zn2+-sensitive soluble exopolyphosphatase from Trypanosoma cruzi depletes polyphosphate and affects osmoregulation.
We report the cloning, expression, purification, and characterization of the Trypanosoma cruzi exopolyphosphatase (TcPPX). The product of this gene (TcPPX), has 383 amino acids and a molecular mass of 43.1 kDa. TcPPX differs from most exopolyphosphatases in its preference for short-chain polyphosphate (poly P). Heterologous expression of TcPPX in Escherichia coli produced a functional enzyme that had a neutral optimum pH and was dramatically inhibited by low concentrations of Zn2+, high concentrations of basic amino acids (lysine and arginine), and heparin. TcPPX is a processive enzyme and does not hydrolyze ATP, pyrophosphate, or p-nitrophenyl phosphate, although it hydrolyzes guanosine 5'-tetraphosphate very efficiently. Overexpression of TcPPX resulted in a dramatic decrease in total short-chain poly P and partial decrease in long-chain poly P. This was accompanied by a delayed regulatory volume decrease after hyposmotic stress. These results support the role of poly P in T. cruzi osmoregulation.
Fang
Jianmin
J
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Paul D. Coverdell Biomedical and Health Sciences Center, Athens, Georgia 30602, USA.
Ruiz
Felix A
FA
Docampo
Melissa
M
Luo
Shuhong
S
Rodrigues
Juliany C F
JC
Motta
Lucimar S
LS
Rohloff
Peter
P
Docampo
Roberto
R
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D011122:Polyphosphates / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014882:Water-Electrolyte Balance; D015032:Zinc / Q000378:metabolism*