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Outer membrane lipoprotein e (P4) of Haemophilus influenzae is a novel phosphomonoesterase
T J Reilly1, D L Chance, A L Smith
1Department of Molecular Microbiology and Immunology, University of Missouri Medical School, Columbia, Missouri 65212, USA.
Abstract:
Haemophilus influenzae exists as a commensal of the upper respiratory tract of humans but also causes infections of contiguous structures. We describe the identification, localization, purification, and characterization of a novel, surface-localized phosphomonoesterase from a nontypeable H. influenzae strain, R2866. Sequences obtained from two CNBr-derived fragments of this protein matched lipoprotein e (P4) within the H. influenzae sequence database. Escherichia coli DH5alpha transformed with plasmids containing the H. influenzae hel gene, which encodes lipoprotein e (P4), produced high levels of a membrane-associated phosphomonoesterase. The isolated approximately 28-kDa enzyme was tartrate resistant and displayed narrow substrate specificity with the highest activity for arylphosphates, excluding 5-bromo-4-chloro-3-indolylphosphate. Optimum enzymatic activity was observed at pH 5.0 and only in the presence of divalent copper. The enzyme was inhibited by vanadate, molybdate, and EDTA but was resistant to inorganic phosphate. The association of phosphomonoesterase activity with a protein that has also been recognized as a heme transporter suggests a unique role for this unusual phosphohydrolase.
Insights
Researchers identified a novel surface enzyme in Haemophilus influenzae, a common bacterium. This phosphomonoesterase, linked to a heme transporter, may play a unique role in bacterial infections.
Area of Science:
- Microbiology
- Enzymology
- Bacterial Pathogenesis
Background:
- Haemophilus influenzae is a human commensal and opportunistic pathogen.
- Surface-localized enzymes play critical roles in bacterial interactions and virulence.
Purpose of the Study:
- To identify, purify, and characterize a novel surface-localized phosphomonoesterase from nontypeable H. influenzae.
- To elucidate the enzymatic properties and potential function of this novel enzyme.
Main Methods:
- Protein purification and characterization.
- Gene cloning and expression in Escherichia coli.
- Enzymatic activity assays under various conditions.
Main Results:
- A novel, approximately 28-kDa surface phosphomonoesterase was identified and purified.
- The enzyme, encoded by the hel gene (lipoprotein e/P4), showed optimal activity at pH 5.0 with divalent copper.
- The enzyme exhibited tartrate resistance and specific inhibition by vanadate, molybdate, and EDTA.
Conclusions:
- The identified phosphomonoesterase is associated with lipoprotein e (P4) in H. influenzae.
- Its dual role as a phosphohydrolase and potential heme transporter suggests a unique function in bacterial physiology or pathogenesis.