A secondary kinetic isotope effect study of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase-catalyzed reaction: evidence for a retroaldol-aldol rearrangement.
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.
Munos
Jeffrey W
JW
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Pu
Xiaotao
X
Mansoorabadi
Steven O
SO
Kim
Hak Joong
HJ
Liu
Hung-wen
HW
D000438:Alcohols / Q000138:chemical synthesis* / Q000737:chemistry; D000447:Aldehydes / Q000138:chemical synthesis / Q000737:chemistry*; D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D007659:Ketones / Q000737:chemistry*; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism
D000438:Alcohols; D000447:Aldehydes; D007659:Ketones; D009097:Multienzyme Complexes; D010088:Oxidoreductases; C114231:1-deoxy-D-xylulose 5-phosphate reductoisomerase; D019747:Aldose-Ketose Isomerases