Format

Send to:

Choose Destination
See comment in PubMed Commons below
Mol Biosyst. 2009 Sep;5(9):935-44. doi: 10.1039/b903513c. Epub 2009 May 15.

Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using (1)H-(31)P-(31)P correlation NMR spectroscopy.

Author information

  • 1Biomolecular NMR Center, The Johns Hopkins University, Baltimore, MD 21218, USA. ananya@jhu.edu

Abstract

The biogenesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) is accomplished by the methylerythritol phosphate (MEP) pathway in plants, bacteria and parasites, making it a potential target for the development of anti-infective agents and herbicides. The biosynthetic enzymes comprising this pathway catalyze intriguing chemical transformations on diphosphate scaffolds, offering an opportunity to generate novel analogs in this synthetically challenging compound class. Such a biosynthetic approach to generating new diphosphate analogs may involve transformation through discrete diphosphate species, presenting unique challenges in structure determination and characterization of unnatural enzyme-generated diphosphate products produced in tandem. We have developed (1)H-(31)P-(31)P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 microM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the (31)P nucleus with the high intrinsic sensitivity of proton NMR, (1)H-(31)P-(31)P correlation spectroscopy is particularly useful for characterization of unnatural diphosphate enzyme products in the MEP pathway. As proof of principle, we demonstrate the rapid characterization of natural enzyme products of the enzymes IspD, E and F in tandem enzyme incubations. In addition, we have characterized several unnatural enzyme products using this technique, including new products of cytidyltransferase IspD bearing erythritol, glycerol and ribose components. The results of this study indicate that IspD may be a useful biocatalyst and highlight (1)H-(31)P-(31)P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.

PMID:
19668858
[PubMed - indexed for MEDLINE]
PMCID:
PMC3161243
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Royal Society of Chemistry Icon for PubMed Central
    Loading ...
    Write to the Help Desk