M. Durairaj et al., GENETIC AND BIOCHEMICAL-ANALYSIS OF THE CYSTEINYL RESIDUES OF ISOPENICILLIN N SYNTHASE FROM STREPTOMYCES-CLAVULIGERUS, Canadian journal of microbiology, 42(8), 1996, pp. 870-875
Isopenicillin N synthase (IPNS) from Streptomyces clavuligerus catalys
es the oxidative cyclization of the acyclic tripeptide delta-(L-alpha-
aminoadipyl)-L-cysteinyl-D-valine into isopenicillin N. All four of th
e cysteine residues found in this enzyme were mutated individually int
o serine residues, either by the polymerase chain reaction or by singl
e-strand site-directed mutagenesis. Functional analysis of these singl
e mutants showed that the C104S mutant lost more than 96% of its activ
ity, while the remaining C37S, C142S, and C251S mutants each lost 30-5
0% of their activity. Treatment with the thiol-group-specific reagent
N-ethylmaleimide confirmed the importance of the cysteine 104 residue.
Activity analysis of an IPNS triple mutant (C37S, C142S, and C251S),
prepared by recombining fragments of the IPNS-encoding pcbC gene from
each of the three single mutants, showed that it had lost more than 90
% of its activity. Conformational analysis by circular dichroism spect
roscopy indicated that the IPNS triple mutant was structurally differe
nt from the wild type, suggesting that the loss of activity may be due
to conformational changes rather than active site modifications.