Absence of substrate channeling between active sites in the Agrobacterium tumefaciens IspDF and IspE enzymes of the methyl erythritol phosphate pathway

Biochemistry
Christian LherbetC Dale Poulter

Abstract

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 ...Continue Reading

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Citations

Jul 31, 2012·ACS Chemical Biology·J Kipchirchir Bitok, Caren Freel Meyers
Jul 12, 2011·Cellular and Molecular Life Sciences : CMLS·Tobias GräwertWolfgang Eisenreich
Feb 19, 2013·Plant Science : an International Journal of Experimental Plant Biology·Andréa Hemmerlin
Feb 11, 2014·Photosynthesis Research·Fiona K DaviesMatthew C Posewitz
Jul 28, 2016·Protein Science : a Publication of the Protein Society·Yi LinMartin St Maurice
Dec 21, 2016·Chemical Reviews·Annika Frank, Michael Groll
Jul 12, 2014·Journal of Experimental Botany·Montserrat SaladiéMichael A Phillips
Apr 20, 2007·The Journal of Biological Chemistry·William N Hunter

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