A secondary kinetic isotope effect study of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement

J Am Chem Soc. 2009 Feb 18;131(6):2048-9. doi: 10.1021/ja807987h.

Abstract

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR, also known as methyl-d-erythritol 4-phosphate (MEP) synthase) is a NADPH-dependent enzyme, which catalyzes the conversion of DXP to MEP in the nonmevalonate pathway of isoprene biosynthesis. Two mechanisms have been proposed for the DXR-catalyzed reaction. In the alpha-ketol rearrangement mechanism, the reaction begins with deprotonation of the C-3 hydroxyl group followed by a 1,2-migration to give methylerythrose phosphate, which is then reduced to MEP by NADPH. In the retroaldol/aldol rearrangement mechanism, DXR first cleaves the C3-C4 bond of DXP in a retroaldol manner to generate a three-carbon and a two-carbon phosphate bimolecular intermediate. These two species are then reunited by an aldol reaction to form a new C-C bond, yielding an aldehyde intermediate. Subsequent reduction by NADPH affords MEP. To differentiate these mechanisms, we have prepared [3-(2)H]- and [4-(2)H]-DXP and carried out a competitive secondary kinetic isotope effect (KIE) study of the DXR reaction. The normal 2 degrees KIEs observed for [3-(2)H]- and [4-(2)H]-DXP provide compelling evidence supporting a retroaldol/aldol mechanism for the rearrangement catalyzed by DXR, with the rate-limiting step being cleavage of the C3-C4 bond of DXP.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohols / chemical synthesis*
  • Alcohols / chemistry
  • Aldehydes / chemical synthesis
  • Aldehydes / chemistry*
  • Aldose-Ketose Isomerases / chemistry*
  • Aldose-Ketose Isomerases / metabolism
  • Catalysis
  • Ketones / chemistry*
  • Kinetics
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / metabolism
  • Oxidoreductases / chemistry*
  • Oxidoreductases / metabolism

Substances

  • Alcohols
  • Aldehydes
  • Ketones
  • Multienzyme Complexes
  • Oxidoreductases
  • 1-deoxy-D-xylulose 5-phosphate reductoisomerase
  • Aldose-Ketose Isomerases