Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein

Abstract

Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with 13 C-labeled 1-deoxy-D-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. 13 C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.isoprenoid biosynthesis ͉ 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate

Possible direct involvement of the active‐site [4Fe–4S] cluster of the GcpE enzyme from Thermus thermophilus in the conversion of MEcPP

“…Based on the similarity of the EPR signals in GcpE and FTR we propose that the transient signal detected in GcpE is due to binding and stabilization of a radical-type intermediate by the 4Fe cluster. This is an important finding since several mechanisms have been proposed in the literature [10,13,16,33], but in only two papers was a direct role proposed for the 4Fe cluster [11,34].…”

“…This procedure, however, can result in the presence of a mix of clusters types. Sequence alignments of the available GcpE sequences [13][14][15][16][17] show that there are only three highly conserved cysteine residues that in principle can coordinate a [4Fe-4S] cluster that contains a unique Fe atom or a [3Fe-4S] cluster. In the case of enzyme from Thermosynechococcus elongatus the reconstitution procedure resulted in an enzyme preparation that showed an absorption spectrum with bands at 305, 395 and 585 nm, which was interpreted as being due to a [4Fe-4S] + cluster [12].…”

Disruption of the 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) gene results in albino, dwarf and defects in trichome initiation and stomata closure in Arabidopsis

“…Then, DXP is transformed to MEP, catalyzed by DXR [14]. Finally, MEP is converted to IPP and DMAPP after consecutive steps catalyzed by 2-C-methyl-d-erythritol-4-phosphate cytidylyltransferase (CMS), 4-(cytidine 5′-diphospho)-2-C-methyl-d-erythritol kinase (CMK), 2-C-methyl-derythritol-2,4-cyclodiphosphate synthase (MCS), 4-hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) and isopentenyl diphosphate isomerase (IPI) [9,5,15,16].…”

Arabidopsis CYP735A1 and CYP735A2 Encode Cytokinin Hydroxylases That Catalyze the Biosynthesis of trans-Zeatin

“…The synthesis of tZR 5Ј-monophosphate (tZRMP) from AMP and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate (HMBDP), a likely precursor, has been demonstrated to be catalyzed by TZS, an agrobacterial IPT (14). However, HMBDP was identified as a metabolic intermediate of the methylerythritol phosphate (MEP) pathway (15), which occurs in bacteria and plastids (16). Selective labeling experiments using 13 Clabeled precursors specific for either the MEP or MVA pathway demonstrated that the isoprenoid side chain of iP and tZ predominantly originates from the MEP pathway, whereas a large fraction of the cZ side chain is derived from the MVA pathway (17).…”