Characterization and Mechanistic Studies of Type II Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus†

William Kittleman, Christopher J. Thibodeaux, Yung-nan Liu, Hua Zhang, and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712
Biochemistry, 2007, 46 (28), pp 8401–8413
DOI: 10.1021/bi700286a
Publication Date (Web): June 22, 2007
Copyright © 2007 American Chemical Society
†

 This work was supported in part by a Welch Foundation Grant (F-1511) and a National Institutes of Health Grant (GM40541).

,
*

 To whom correspondence and reprint requests should be addressed. Phone:  512-232-7811. Fax:  512-471-2746. E-mail:  h.w.liu@mail.utexas.edu.

Abstract

Abstract Image

The recently identified type II isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase (IDI-2) is a flavoenzyme that requires FMN and NAD(P)H for activity. IDI-2 is an essential enzyme for the biosynthesis of isoprenoids in several pathogenic bacteria including Staphylococcus aureus, Streptococcus pneumoniae, and Enterococcus faecalis, and thus is considered as a potential new drug target to battle bacterial infections. One notable feature of the IDI-2 reaction is that there is no net change in redox state between the substrate (IPP) and product (DMAPP), indicating that the FMN cofactor must start and finish each catalytic cycle in the same redox state. Here, we report the characterization and initial mechanistic studies of the S. aureus IDI-2. The steady-state kinetic analyses under aerobic and anaerobic conditions show that FMN must be reduced to be catalytically active and the overall IDI-2 reaction is O2-sensitive. Interestingly, our results demonstrate that NADPH is needed only in catalytic amounts to activate the enzyme for multiple turnovers of IPP to DMAPP. The hydride transfer from NAD(P)H to reduce FMN is determined to be pro-S stereospecific. Photoreduction and oxidation−reduction potential studies reveal that the S. aureus IDI-2 can stabilize significant amounts of the neutral FMN semiquinone. In addition, reconstitution of apo-IDI-2 with 5-deazaFMN resulted in a dead enzyme, whereas reconstitution with 1-deazaFMN led to the full recovery of enzyme activity. Taken together, these studies appear to support a catalytic mechanism in which the reduced flavin coenzyme mediates a single electron transfer to and from the IPP substrate during catalysis.

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History

  • Published In Issue July 17, 2007
  • Received February 9, 2007
    Revised Manuscript Received May 3, 2007

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