• Journal of Applied Biological Chemistry
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• v.48 no.4
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• pp.183-186
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• 2005

Arabidopsis AHL gene encodes a 3'(2')-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase that plays a role in the sulfate activation pathway. We complemented E. coli cysQ mutant defective in cysteine biosynthesis with the AHL gene. AHL cDNA was cloned into the prokaryotic expression vector pKK388-1 and transformed into the bacterial mutant. Since cysQ mutant is a leaky cysteine auxotroph only under aerobic conditions, the bacteria were grown in liquid media with vigorous shaking to provide more aeration. In cysteine-free medium, cysQ mutant and the mutant harboring empty vector did not grow well, whereas cells harboring AHL cDNA exhibited significantly improved growth with doubling time of approximately 3 h. cysQ is known to encode a 3'(2'),5'-diphosphonucleoside 3'(2')-phosphohydrolase (DPNPase). However, our data suggest that cysQ protein has PAP-specific phosphatase activity in addition to DPNPase activity. Microbial complementation procedure described in this paper is useful for structure-activity studies of PAP-specific phosphatases identified from microbes and plants.

• Journal of Applied Biological Chemistry
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• v.42 no.4
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• pp.169-174
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• 1999

Arabidopsis AHL gene contains 4 exons encoding a putative protein highly homologous to the yeast salt-sensitive enzyme HAL2, a 3'(2'),5'-bisphosphate nucleotidase involving in reductive sulfate assimilation. AHL cDNA complemented yeast met22 (hal2) mutant. AHL fusion protein expressed in E. coli exhibited $Mg^{2+}$-dependent, 3'-phosphoadenosine 5'-phosphate (PAP)-specific phosphatase activity. $Li^+,\;Na^+,\;K^+$ and $Ca^{2+}$ ions inhibit the enzyme activity by competing with $Mg^{2+}$ for the active site of the enzyme. The enzyme activity was also sensitive to ${\mu}M$ concentrations of toxic heavy metal ions such as $Cd^{2+},\;Cu^{2+}$ and $Zn^{2+}$, but was not recovered by addition of more $Mg^{2+}$ ions, suggesting that these ions inactivate the enzyme with a mechanism other than competition with $Mg^{2+}$ ions. Inhibition of the AHL enzyme activity may result in accumulation of PAP, which is highly toxic to the cell. Thus, the AHL enzyme could be one of the intial targets of heavy metal toxicity in plants.