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 g rowth. The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chrom otography on DEAE-Sephacel and Cibacron Blue 3GA-agarose. The active e nzyme consists of a single polypeptide with a mass of 34 kDa. At 25 de grees C, it has a pH optimum of 8.25, a K-m for cAMP of 73 mu m, and a V-max of 3700 mu mol of cAMP hydrolyzed/min/mg protein (turnover numb er of 1.24 x 10(5)/min). The specific activity of the V. fischeri enzy me is approximately 20-fold greater than that of any previously charac terized CNP when comparisons of activity are made at the same assay te mperature. Activity increases with temperature up to 60 degrees C. The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, an d calcium can restore activity of the apoenzyme 60 varying degrees. Th e exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacter ium to scavenge 3':5'-cyclic nucleotides in seawater and that the enzy me plays a role in cAMP-mediated host-symbiont interactions.