Characterization of a Unique Class C Acid Phosphatase from Clostridium perfringens

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 ϳ31 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 mol 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.Clostridium perfringens is a ubiquitous gram-positive, endospore-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 infections in humans and animals. As a species, C. perfringens is a very heterogeneous group of organisms with respect to metabolic 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. perfringens-encoded toxins, as well as molecular and morphological 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 investigators (1, 7) has recognized the clinical utility of acid phosphatase 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 ϳ95% 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 essential roles in the generation, acquisition, and mobilization of inorganic phosphate and critical roles in phosphoryl relay systems intimately involved in signal transduction pathways in both prokaryotes and eukaryotes. A subgroup of phosphatases has been designated nonspecific acid phosphatases (NSAPs) by Thaller and coworkers (45) and includes those bacterial polyspecific phosphatases that are secreted across the cytoplasmic membrane and exhibit optimum catalytic activity at an acidic pH (41). NSAPs are recognized as a distinct group of phosphatases and categorized into three classes (46). The class C enzyme...

Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

“…For example, we have shown here that the catalytic efficiency of rP4 for 5′-AMP is approximately half that of NMN and only twice that of 3′-AMP. Similar results have been reported for other CCAPs 15,17,18…”

“…In addition to e (P4),1,2,12–14 several CCAPs have been characterized to various degrees, including those from Elizabethkingia meningosepticum (OlpA15), Streptococcus equisimilis (LppC16), Helicobacter pylori (HppA17), and Clostridium perfringens 18,19. CCAPs are dimeric enzymes that exhibit phosphomonoesterase activity for commonly used aryl phosphate substrates such as p -nitrophenyl phosphate ( p NPP) and 4-methylumbelliferyl phosphate.…”

“…CCAPs do not exhibit a strong base preference among this class of substrate. OlpA, LppC,20 rP4 ( vide infra ) and the C. perfringens enzyme18 also exhibit activity with nucleoside 3′-monophosphates, but with lower efficiency than nucleoside 5′-monophosphate substrates. Thus, the available in vitro data suggest that CCAPs function primarily as nonspecific 5′-, 3′-nucleotidases.…”

Crystal structure and immunogenicity of the class C acid phosphatase from Pasteurella multocida

“…The best-fit value of the molecular weight is 60.1 ± 0.2 kDa, which is within 3 % of the theoretical molecular weight of a dimer (58.1 kDa). This result is consistent with sedimentation and size exclusion chromatography data showing that rP4 and the CCAPs from Helicobacter pylori and Clostridium perfringens are dimeric in solution [6, 12, 13]. …”

Histochemical Imaging of Alkaline Phosphatase Using a Novel Fluorescent Substrate

“…[1][2][3][4][5] However, these substrates are not used for histochemical staining, because of their solubility in water after phosphatase reaction. 5-Bromo-4-chloro-3-indolyl-phosphate (X-Phos) has been mainly used for histochemical detection of phosphatase activity.…”