Purification, crystallization and characterization of N-acetylneuraminate lyase from Escherichia coli.
N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.
Aisaka
K
K
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd., Japan.
Igarashi
A
A
Yamaguchi
K
K
Uwajima
T
T
D000595:Amino Acid Sequence; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003460:Crystallization; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D006882:Hydroxyapatites; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation
D006882:Hydroxyapatites; D017886:Durapatite; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase