Structure-function studies of the non-heme iron active site of isopenicillin N synthase: Some implications for catalysis

Rachel Kreisberg-Zakarin, Ilya Borovok, Michaela Yanko, Felix Frolow, Yair Aharonowitz, Gerald Cohen*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygenase that catalyzes the ring closure of δ-(L-α-aminoadipoyl)-L- cysteinyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, dioxygen and H2O. We previously showed by site-directed mutagenesis that residues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis are essential for activity and correspond to the iron ligands identified by crystallography. To evaluate the importance of the nature of the protein ligands for activity, His214 and His268 were exchanged with asparagine, aspartic acid and glutamine, and Asp214 replaced with glutamic acid, histidine and cysteine, each of which has the potential to bind iron. Only the Asp214Glu mutant retained activity, ~ 1% that of the wild type. To determine the importance of the spatial arrangement of the protein ligands for activity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS activity depends critically on the presence of two histidine and one carboxylate ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS crystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination. (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)109-118
Number of pages10
JournalBiophysical Chemistry
Volume86
Issue number2-3
DOIs
StatePublished - 30 Aug 2000

Keywords

  • Active-site protein ligands
  • Isopenicillin N synthase
  • Mutagenesis
  • Non-heme iron dioxygenase
  • Streptomyces

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