Structural studies on the reaction of isopenicillin N synthase with the truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine.
Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.
Long
Alexandra J
AJ
Chemistry Research Laboratory, University of Oxford, UK.
Clifton
Ian J
IJ
Roach
Peter L
PL
Baldwin
Jack E
JE
Rutledge
Peter J
PJ
Schofield
Christopher J
CJ
D000409:Alanine / Q000737:chemistry*; D000693:Anaerobiosis; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D032842:Emericella / Q000201:enzymology; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000737:chemistry*; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010406:Penicillins / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
D005296:Ferrous Compounds; D009842:Oligopeptides; D010406:Penicillins; C094178:aminoadipoyl-cysteinyl-allylglycine; D009569:Nitric Oxide; D010088:Oxidoreductases; C038370:isopenicillin N synthetase; D000409:Alanine; D005998:Glycine