Cloning, expression and characterization of an extracellular enolase from Leuconostoc mesenteroides.
Enolase on the surface of streptococci putatively facilitates pathogenic invasion of the host organisms. The related Leuconostoc mesenteroides 512FMCM is nonpathogenic, but it too has an extracellular enolase. Purified isolates of extracellular dextransucrase from cultures of L. mesenteroides contain minute amounts of enolase, which separate as small crystals. Expression of L. mesenteroides enolase in Escherichia coli provides a protein (calculated subunit mass of 47 546 Da) catalyzing the conversion of 2-phsopho-D-glycerate to phosphoenolpyruvate. The pH optimum is 6.8, with Km and kcat values of 2.61 mM and 27.5 s(-1), respectively. At phosphate concentrations of 1 mM and below, fluoride is a noncompetitive inhibitor with respect to 2-phospho-D-glycerate, but in the presence of 20 mM phosphate, fluoride becomes a competitive inhibitor. Recombinant enolase significantly inhibits the activity of purified dextransucrase, and does not bind human plasminogen. Results here suggest that in some organisms enolase may participate in protein interactions that have no direct relevance to pathogenic invasion.
Lee
Jin-Ha
JH
Engineering Research Institute, Chonnam National University, Gwang-Ju, South Korea.
Kang
Hee-Kyoung
HK
Moon
Young-Hwan
YH
Cho
Dong Lyun
DL
Kim
Doman
D
Choe
Jun-Yong
JY
Honzatko
R
R
Robyt
John F
JF
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004269:DNA, Bacterial / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010751:Phosphopyruvate Hydratase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase; D010751:Phosphopyruvate Hydratase