Substrate specificity of isopenicillin N synthase.
Highly purified isopenicillin N synthase (IPNS) from two sources (naturally occurring in Penicillium chrysogenum and that expressed in Escherichia coli via a cloned gene derived from Cephalosporium acremonium) have been isolated and utilized in vitro to test synthetic modifications of the natural substrate, (L-alpha-amino-delta-adipyl)-L-cysteinyl-D-valine (ACV). A very sensitive procedure utilizing the ability of beta-lactams to induce the synthesis of beta-lactamase was employed to determine whether an ACV analogue could serve as a substrate for IPNS. A wide variety of amino and carboxyl terminal tripeptide substitutions were examined and found to elicit positive beta-lactamase induction profiles. However, none of these modifications were found to function as efficiently as a substrate as ACV. One of the beta-lactam products which was formed from the reaction of IPNS and the tripeptide analogue was independently synthesized and evaluated for antibacterial activity. Modification of the L-cysteine residue in the second position of ACV resulted in tripeptides that were unable to serve as substrates. Conversion of the D-valine residue in the third position of ACV to an aromatic amino acid or to a highly electronegative residue such as trifluorovaline resulted in elimination of substrate activity and creation of an inhibitor of the enzyme.
Huffman
G W
GW
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285.
Gesellchen
P D
PD
Turner
J R
JR
Rothenberger
R B
RB
Osborne
H E
HE
Miller
F D
FD
Chapman
J L
JL
Queener
S W
SW
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001412:Bacillus subtilis; D016982:Micrococcus luteus; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011549:Pseudomonas; D013379:Substrate Specificity
D000900:Anti-Bacterial Agents; D009842:Oligopeptides; C011638:5-(2-aminoadipyl)cysteinylvaline; D010088:Oxidoreductases; C038370:isopenicillin N synthetase