Purification and characterization of the isopenicillin N synthase of Streptomyces lactamdurans.
The isopenicillin N synthase (cyclase) of Streptomyces lactamdurans (syn. Nocardia lactamdurans) has been purified to near homogeneity as judged by SDS-PAGE and isoelectric focusing. This enzyme catalyses the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The enzyme required DTT, Fe2+ and oxygen and it was greatly stimulated by ascorbic acid. It was strongly inhibited by Co2+, Zn2+ and Mn2+. Optimal pH and temperature were 7.0 and 25 degrees C (with the assay conditions used), respectively. The apparent Km of isopenicillin N synthase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.18 mM. The enzyme is a monomer with an Mr of 26,500 +/- 1000 and a pI of 6.55.
Castro
J M
JM
Departamento de Microbiologia, Facultad de Biologia, Universidad de León, Spain.
Liras
P
P
Laíz
L
L
Cortés
J
J
Martín
J F
JF
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D010088:Oxidoreductases; D013302:Streptomyces / Q000201:enzymology*
D004798:Enzymes; D010088:Oxidoreductases; C038370:isopenicillin N synthetase