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Characterization of a unique class C acid phosphatase from Clostridium perfringens
Thomas J Reilly1, Deborah L Chance, Michael J Calcutt
1Department of Veterinary Pathobiology, University of Missouri, Columbia, 65211, USA. reillyt@missouri.edu
Abstract:
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 approximately 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 K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/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.
Insights
This study characterizes the acid phosphatase enzyme from Clostridium perfringens, a medically important anaerobe. Understanding this enzyme aids in diagnosing C. perfringens infections.
Area of Science:
- Microbiology
- Enzymology
- Bacterial Pathogenesis
Background:
- Clostridium perfringens is a significant Gram-positive anaerobic pathogen.
- Acid phosphatase activity is a key diagnostic marker for C. perfringens.
- Characterization of this specific enzyme was previously unreported.
Purpose of the Study:
- To characterize the soluble recombinant acid phosphatase from C. perfringens.
- To understand the enzyme's biochemical properties and substrate specificity.
- To investigate its role in C. perfringens identification.
Main Methods:
- Expression and purification of a soluble recombinant acid phosphatase.
- Enzyme activity assays with various substrates at pH 6.
- Determination of kinetic parameters (Km, Vmax) and effects of divalent cations.
- Detection of wild-type enzyme in clinical C. perfringens isolates.
Main Results:
- The denatured enzyme is approximately 31 kDa and a homodimer in solution.
- The enzyme efficiently hydrolyzes para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and nucleoside monophosphates.
- Kinetic parameters indicate substrate affinity (Km 0.2-0.6 mM) and catalytic efficiency (Vmax 0.8-1.6 micromol P(i)/s/mg).
- Enzyme activity is modulated by divalent cations; wild-type enzyme is cell-associated in clinical isolates.
Conclusions:
- The characterized acid phosphatase belongs to the nonspecific acid phosphatase class C.
- It lacks the typical lipid modification of this class.
- This enzyme is a reliable marker for C. perfringens detection in clinical samples.
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