Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway.
The conversion of 2-C-methyl-d-erythritol 4-phosphate (MEP) to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) in the MEP entry into the isoprenoid biosynthetic pathway occurs in three consecutive steps catalyzed by the IspD, IspE, and IspF enzymes, respectively. In Agrobacterium tumefaciens the ispD and ispF genes are fused to encode a bifunctional enzyme that catalyzes the first (synthesis of 4-diphosphocytidyl-2-C-methyl d-erythritol) and third (synthesis of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate) steps. Sedimentation velocity experiments indicate that the bifunctional IspDF enzyme and the IspE protein associate in solution, raising the possibility of substrate channeling among the active sites in these two proteins. Kinetic evidence for substrate channeling was sought by measuring the time courses for product formation during incubations of MEP, CTP, and ATP with the IspDF and IspE proteins with and without an excess of the inactive IspE(D152A) mutant in the presence or absence of 30% (v/v) glycerol. The time dependencies indicate that the enzyme-generated intermediates are not transferred from the IspD active site in IspDF to the active site of IspE or from the active site in IspE to the active site of the IspF module of IspDF.
Lherbet
Christian
C
Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, USA.
Pojer
Florence
F
Richard
Stéphane B
SB
Noel
Joseph P
JP
Poulter
C D
CD
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology*; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013403:Sugar Phosphates / Q000378:metabolism*; D013997:Time Factors; D014461:Ultracentrifugation
C114232:2-C-methylerythritol 4-phosphate; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C443879:methyl-D-erythritol 2,4-cyclodiphosphate; D004896:Erythritol