title: Cloning and expression of IspDF from Mesorhizobium loti. Characterization of a bifunctional protein that catalyzes non-consecutive steps in the methylerythritol phosphate pathway.
abstract: Gram-negative bacteria, plant chloroplasts, green algae and some Gram-positive bacteria utilize the 2-C-methyl-d-erythritol phosphate (MEP) pathway for the biosynthesis of isoprenoids. IspD, ispE, and ispF encode the enzymes required to convert MEP to 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (cMEDP) during the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate in the MEP pathway. Upon analysis of the Mesorhizobium loti genome, ORF mll0395 showed homology to both ispD and ispF and appeared to encode a fusion protein. M. loti ispE was located elsewhere on the chromosome. Purified recombinant IspDF protein was mostly a homodimer, MW approximately 46 kDa/subunit. Incubation of IspDF with MEP, CTP, and ATP gave 4-diphosphocytidyl-2-C-methyl-d-erythritol (CDP-ME) as the only product. When Escherichia coli IspE protein was added to the incubation mixture, cMEDP was formed. In addition, M. loti ORF mll0395 complements lethal disruptions in both ispD and ispF in Salmonella typhimurium. These results indicate that IspDF is a bifunctional protein, which catalyzes the first and third steps in the conversion of MEP to cMEDP.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City, 84112, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Testa
authors.forename: Christian
authors.initials: C
authors.lastname: Lherbet
authors.forename: Florence
authors.initials: F
authors.lastname: Pojer
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004896:Erythritol / Q000031:analogs & derivatives* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013403:Sugar Phosphates / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D013403:Sugar Phosphates; D013729:Terpenes; C443879:methyl-D-erythritol 2,4-cyclodiphosphate; D004896:Erythritol