Skip Navigation


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
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
142/4/533    most recent
mvm162v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Request Permissions
Google Scholar
Right arrow Articles by Matsumura, Y.
Right arrow Articles by Sagami, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Matsumura, Y.
Right arrow Articles by Sagami, H.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© 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


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?




Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.