ABSTRACT
Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate. Analysis of the 1.97 A crystal structure of the inactive C67A mutant of E. coli isopentenyl diphosphate:dimethylallyl diphosphate isomerase complexed with the mechanism-based inactivator 3,4-epoxy-3-methyl-1-butyl diphosphate is in agreement with an isomerization mechanism involving Glu 116, Tyr 104, and Cys 67. In particular, the results are consistent with a mechanism where Glu116 is involved in the protonation step and Cys67 in the elimination step.
Subject(s)
Carbon-Carbon Double Bond Isomerases/antagonists & inhibitors , Carbon-Carbon Double Bond Isomerases/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli/enzymology , Point Mutation/genetics , Binding Sites , Carbon-Carbon Double Bond Isomerases/genetics , Carbon-Carbon Double Bond Isomerases/metabolism , Crystallography, X-Ray , Enzyme Inhibitors/pharmacology , Epoxy Compounds/chemistry , Epoxy Compounds/metabolism , Epoxy Compounds/pharmacology , Escherichia coli/genetics , Hemiterpenes , Isomerism , Organophosphorus Compounds/chemistry , Organophosphorus Compounds/metabolism , Organophosphorus Compounds/pharmacology , ProtonsABSTRACT
Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 A) of complexes of Escherichia coli IPP.DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction.
Subject(s)
Carbon-Carbon Double Bond Isomerases/chemistry , Carbon-Carbon Double Bond Isomerases/metabolism , Escherichia coli/enzymology , Carbon-Carbon Double Bond Isomerases/antagonists & inhibitors , Catalytic Domain , Crystallography , Cysteine/chemistry , Epoxy Compounds/pharmacology , Glutamic Acid/chemistry , Hemiterpenes , Kinetics , Organophosphorus Compounds/pharmacology , Protein Structure, Secondary , Tyrosine/chemistryABSTRACT
Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.
