Gene Cloning, purification, and characterization of a phosphodiesterase from Delftia acidovorans.
A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65 degrees C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K(m) of 2.9 +/- 0.1 mM and a k(cat) of 879 +/- 73 min(-1). The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.
Tehara
Sundiep K
SK
Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720-1462, USA.
Keasling
Jay D
JD
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020562:Betaproteobacteria / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D009596:Nitrophenols; D011994:Recombinant Proteins; C002887:bis(4-nitrophenyl)phosphate; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases