The carboxyl-terminal region of the geranylgeranyl diphosphate synthase is indispensable for the stabilization of the region involved in substrate binding and catalysis.
Rat geranylgeranyl diphosphate synthase (GGPS) and its deletion mutants from the carboxyl terminus were analysed using Escherichia coli harbouring pACYC-crtIB, which contains crtI and crtB encoding the carotenoid biosynthetic enzymes. Mutants (delta-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (delta-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (delta-4, -8, -12 and -16) were overexpressed in E. coli BL21 (DE3) and purified in a stable form by nickel affinity chromatography except for one mutant (delta-16). The farnesyl-transferring activities of wild-type GGPS, delta-4, -8 and -12 mutants were relatively in a ratio of 1.0, 0.84, 0.26 and 0.0015. Each Km value of the four recombinants were estimated to be 0.71, 2.0 2.8 and 55 microM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 microM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that delta-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and delta-4 mutant. These results suggest that the KMFTEENE residing on the carboxyl-terminal sequence of GGPS stabilizes the active region involved in the substrate binding and catalysis.
Matsumura
Yoshihiro
Y
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan.
Kidokoro
Tomohiro
T
Miyagi
Yukino
Y
Marilingaiah
Navya Rani
NR
Sagami
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism; D004224:Diterpenes / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability / Q000235:genetics; D051231:Farnesyltranstransferase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D045782:Hemiterpenes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D011756:Diphosphates; D004224:Diterpenes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C511282:geranyl diphosphate; C043060:3,3-dimethylallyl pyrophosphate; D051231:Farnesyltranstransferase