Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3 and 5 nucleoside monophosphates at pH 6. Calculated Kms ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 mol of Pi/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class. Clostridium perfringens is a ubiquitous gram-positive, endo- spore-forming anaerobic bacterium. While found in soil, the organism is often a constituent of the intestinal flora and is capable of causing severe gastrointestinal and histotoxic infec- tions in humans and animals. As a species, C. perfringens is a very heterogeneous group of organisms with respect to meta- bolic by-products produced in vitro and pathogenic potential (25). Although the literature is replete with descriptions of the immunological, bio- and physicochemical attributes of C. per- fringens-encoded toxins, as well as molecular and morpholog- ical details of sporulation, detailed analysis of other constituent enzymes, including those involved in acquisition and regulation of inorganic phosphate, has received relatively little attention. This lack of information is notable. A growing cadre of inves- tigators (1, 7) has recognized the clinical utility of acid phos- phatase as a biochemical marker for the identification of C. perfringens isolated from water and other sources and its use in the differentiation of C. perfringens from 95% of all currently recognized clostridial species in the genus (42). Acid phosphatases (EC 3.1.3.2) are ubiquitous and catalyze, at an acidic pH, the transfer of phosphoryl groups from phosphomonoesters to water (48). These enzymes play essen- tial roles in the generation, acquisition, and mobilization of inorganic phosphate and critical roles in phosphoryl relay sys- tems intimately involved in signal transduction pathways in both prokaryotes and eukaryotes. A subgroup of phosphatases