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Journal of Biochemistry Advance Access originally published online on September 10, 2007
Journal of Biochemistry 2007 142(4):533-537; doi:10.1093/jb/mvm162
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© 2007 The Japanese Biochemical Society.

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*

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan

*To whom correspondence should be addressed. Tel: +81-22-217-5622, Fax: +81-22-217-5620, E-mail: yasagami{at}tagen.tohoku.ac.jp

Received June 1, 2007; Accepted August 10, 2007


  Abstract

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 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 {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.

Key Words: carotenoid, deletion mutants, geranylgeranyl diphosphate synthase, prenyltransferase, structure and activity

Abbreviations: DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GPP, geranyl diphosphate; GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; IPP, isopentenyl diphosphate


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