title: Enzymatic and biological characteristics of enolase in Brucella abortus A19.
abstract: Brucella abortus is the etiological agent of brucellosis, a disease causing human public health problems as well as major economic losses in domestic animal industries. In this study, the enolase gene of B. abortus A19 was cloned, sequenced and expressed in Escherichia coli BL21. Bacterial-expressed enolase protein (His-eno) was purified and its ability to catalyze the conversion of 2-phosphoglycerate (2-PGE) to phosphoenolpyruvate (PEP) (hereon referred to as enolase activity) was analyzed. Michaelis constant (K(m)) and maximum reaction velocity (V(max)) of the reaction was determined to be 2.0 × 10(-3) M and 178 μM l(-1)min(-1), respectively. Factors influencing the enolase activity of His-eno, such as pH, the presence of metal ions and temperature were investigated in vitro. The results showed that His-eno exhibited maximal enolase activity in pH 8.5 reaction buffer containing 10 mM MgSO(4) at 37 °C. In addition to studying the enzyme activity, binding assays were performed to provide insights into the function of His-eno on pathogenesis and immunity. His-eno exhibits fibronectin-binding ability in immunoblotting assay, suggesting that enolase may play a role in B. abortus colonization, persistence, and invasion of host tissue. Furthermore, Western blot demonstrated His-eno's binding ability to 34 bovine B. abortus positive sera, suggesting that future studies may find enolase a useful as a diagnostic marker or a vaccine candidate for brucellosis.
authors.affiliation: Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, People's Republic of China.
authors.forename: Xiangan
authors.initials: X
authors.lastname: Han
authors.forename: Chan
authors.initials: C
authors.lastname: Ding
authors.forename: Hongjun
authors.initials: H
authors.lastname: Chen
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Hu
authors.forename: Shengqing
authors.initials: S
authors.lastname: Yu
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002003:Brucella abortus / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005353:Fibronectins / Q000378:metabolism; D005988:Glyceric Acids / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D017931:DNA Primers; D005353:Fibronectins; D005988:Glyceric Acids; D008670:Metals; C008885:2-phosphoglycerate; D010728:Phosphoenolpyruvate; D010751:Phosphopyruvate Hydratase