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Unexpected Oxidation of a Depsipeptide Substrate Analogue in Crystalline Isopenicillin N Synthase
Author(s) -
Daruzzaman Adam,
Clifton Ian J.,
Adlington Robert M.,
Baldwin Jack E.,
Rutledge Peter J.
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500282
Subject(s) - active site , stereochemistry , chemistry , catalytic cycle , steric effects , substrate (aquarium) , oxidoreductase , hydroxymethyl , enzyme , organic chemistry , biology , ecology
Isopenicillin N synthase (IPNS) is a non‐heme iron( ii) ‐dependent oxidase that is central to penicillin biosynthesis. Herein, we report mechanistic studies of the IPNS reaction in the crystalline state, using the substrate analogue δ‐( L ‐α‐aminoadipoyl)‐(3 R )‐methyl‐ L ‐cysteine D ‐α‐hydroxyisovaleryl ester (AmC O V) to probe the early stages of the catalytic cycle. The X‐ray crystal structure of the anaerobic IPNS:Fe II :AmC O V complex was solved to 1.40 Å resolution, and it reveals several subtle differences in the active site relative to the complex of the enzyme with its natural substrate. The crystalline IPNS:Fe II :AmC O V complex was then exposed to oxygen gas at high pressure; this brought about reaction to give what appears to be a hydroxymethyl/ene‐thiol product. A mechanism for this reaction is proposed. These results offer further insight into the delicate interplay of steric and electronic effects in the IPNS active site and the mechanistic intricacies of this remarkable enzyme.