Journal Article

The Carboxyl-terminal Region of the Geranylgeranyl Diphosphate Synthase is Indispensable for the Stabilization of the Region Involved in Substrate Binding and Catalysis

Yoshihiro Matsumura, Tomohiro Kidokoro, Yukino Miyagi, Navya Rani Marilingaiah and Hiroshi Sagami

in The Journal of Biochemistry

Published on behalf of The Japanese Biochemical Society

Volume 142, issue 4, pages 533-537
Published in print October 2007 | ISSN: 0021-924X
Published online October 2007 | e-ISSN: 1756-2651 | DOI: https://dx.doi.org/10.1093/jb/mvm162
The Carboxyl-terminal Region of the Geranylgeranyl Diphosphate Synthase is Indispensable for the Stabilization of the Region Involved in Substrate Binding and Catalysis

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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 (Δ-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (Δ-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (Δ-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 (Δ-16). The farnesyl-transferring activities of wild-type GGPS, Δ-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 μM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 μM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that Δ-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and Δ-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.

Keywords: carotenoid; deletion mutants; geranylgeranyl diphosphate synthase; prenyltransferase; structure and activity

Journal Article.  2805 words.  Illustrated.

Subjects: Biochemistry

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