NDLI: Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy

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

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Molecular BioSystems Vol - 5 Issue - 8

Molecular BioSystems Vol - 5 Issue - 7

Molecular BioSystems Vol - 5 Issue - 6

Molecular BioSystems Vol - 5 Issue - 5

Molecular BioSystems Vol - 5 Issue - 4

Molecular BioSystems Vol - 5 Issue - 3

Molecular BioSystems Vol - 5 Issue - 2

Molecular BioSystems Vol -4

Molecular BioSystems Vol -3

Molecular BioSystems Vol -2

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

Content Provider	Royal Society of Chemistry (RSC)
Author	Bitok, J. Kipchirchir Hassis-LeBeau, Maria E. Shah, Meha H. Meyers, Caren L. Freel Majumdar, Ananya
Copyright Year	2009
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 1H–31P–31P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 μM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the 31P nucleus with the high intrinsic sensitivity of proton NMR, 1H–31P–31P 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 1H–31P–31P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.
Starting Page	935
Ending Page	944
Page Count	10
File Format	HTM / HTML PDF
ISSN	1742206X
Volume Number	5
Issue Number	9
Journal	Molecular BioSystems
DOI	10.1039/b903513c
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Open
Subject Keyword	Dimethylallyl pyrophosphate Terpenoid Natural abundance Enzyme IPP Glycerol Nuclear magnetic resonance spectroscopy Proton nuclear magnetic resonance Erythritol Two-dimensional nuclear magnetic resonance spectroscopy Ribose Phosphate Pyrophosphate Bacteria Non-mevalonate pathway Nuclear magnetic resonance Isopentenyl pyrophosphate
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Biotechnology

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Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy † ‡

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

Structural genomics of enzymes involved in sterol/isoprenoid biosynthesis.

Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis.

Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

De novo fragment-based design of inhibitors of DXS guided by spin-diffusion-based NMR spectroscopy

Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors.

Integrated analysis of isopentenyl pyrophosphate (IPP) toxicity in isoprenoid-producing Escherichia coli

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

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Probing phosphorylation by non-mammalian isoprenoid biosynthetic enzymes using 1H–31P–31P correlation NMR spectroscopy † ‡

Escherichia coli engineered to synthesize isopentenyl diphosphate and dimethylallyl diphosphate from mevalonate: a novel system for the genetic analysis of the 2-C-methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.

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

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