Purification and properties of periplasmic 3':5'-cyclic nucleotide phosphodiesterase. A novel zinc-containing enzyme from the marine symbiotic bacterium Vibrio fischeri.
The 3':5'-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm, allows this symbiotic bacterium to utilize extracellular 3':5'-cyclic nucleotides (e.g. cAMP) as sole sources of carbon and energy, nitrogen, and phosphorus for growth. The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and Cibacron Blue 3GA-agarose. The active enzyme consists of a single polypeptide with a mass of 34 kDa. At 25 degrees C, it has a pH optimum of 8.25, a Km for cAMP of 73 microns, and a Vmax of 3700 mumol of cAMP hydrolyzed/min/mg protein (turnover number of 1.24 x 10(5)/min). The specific activity of the V. fischeri enzyme is approximately 20-fold greater than that of any previously characterized CNP when comparisons of activity are made at the same assay temperature. Activity increases with temperature up to 60 degrees C. The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can restore activity of the apoenzyme to varying degrees. The exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacterium to scavenge 3':5'-cyclic nucleotides in seawater and that the enzyme plays a role in cAMP-mediated host-symbiont interactions.
Callahan
S M
SM
Biology Department, Woods Hole Oceanographic Institution, Massachusetts 02543, USA.
Cornell
N W
NW
Dunlap
P V
PV
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000302:isolation & purification*; D000242:Cyclic AMP / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature; D014733:Vibrio / Q000201:enzymology*; D015032:Zinc / Q000032:analysis*
D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015032:Zinc