Tài liệu Báo cáo khoa học: Manipulation of prenyl chain length determination mechanism of

Manipulation of prenyl chain length determination

mechanism of cis-prenyltransferases

Yugesh Kharel*, Seiji Takahashi*, Satoshi Yamashita and Tanetoshi Koyama

Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan

In the biosynthesis of isoprenoids, which are the most

structurally diverse and abundant among natural prod￾ucts, all carbon skeletons are biosynthesized using

sequential condensation of isopentenyl diphosphate

(IPP, C5) and allylic diphosphates by actions of prenyl

chain elongating enzymes, commonly called prenyl￾transferases. Prenyltransferases can be classified into

two major groups, i.e. trans- and cis-prenyltransferases,

according to the geometry of the prenyl chain units in

the products [1,2]. The reactions catalysed by prenyl￾transferases start by the formation of allylic cation

after the elimination of pyrophosphate ion to form an

allylic prenyl diphosphate, followed by addition of an

IPP with stereospecific removal of a proton at the

2-position. The only difference between the reaction

catalyzed by trans- and cis-prenyltransferase is the

prochirality of the proton, i.e. pro-R for trans￾prenyltransferase and pro-S for cis-prenyltransferase

(Fig. 1a). However, recent molecular analysis and crys￾tal structure determination of Micrococcus luteus B-P

26 undecaprenyl diphosphate (UPP, C55) synthase,

which catalyses cis-condensation to synthesize UPP,

Keywords

chain-length determination mechanism; cis￾prenyltransferase; isoprenoid biosynthesis;

Micrococcus luteus B-P 26; undecaprenyl

diphosphate synthase

Correspondence

T. Koyama, Institute of Multidisciplinary

Research for Advanced Materials, Tohoku

University, Katahira 2-1-1, Aoba-ku, Sendai,

980-8577, Japan

Fax: +81 22 217 5620

Tel:: +81 22 217 5621

E.mail: [email protected]

Note

*Both authors contributed equally to this

work.

(Received 28 October 2005, revised 9

December 2005, accepted 12 December

2005)

doi:10.1111/j.1742-4658.2005.05097.x

The carbon backbones of Z,E-mixed isoprenoids are synthesized by

sequential cis-condensation of isopentenyl diphosphate (IPP) and an allylic

diphosphate through actions of a series of enzymes called cis-prenyltrans￾ferases. Recent molecular analyses of Micrococcus luteus B-P 26 undecapre￾nyl diphosphate (UPP, C55) synthase [Fujihashi M, Zhang Y-W, Higuchi

Y, Li X-Y, Koyama T & Miki K (2001) Proc Natl Acad Sci USA 98,

4337–4342.] showed that not only the primary structure but also the crystal

structure of cis-prenyltransferases were totally different from those of

trans-prenyltransferases. Although many studies on structure–function rela￾tionships of cis-prenyltransferases have been reported, regulation mecha￾nisms for the ultimate prenyl chain length have not yet been elucidated.

We report here that the ultimate chain length of prenyl products can be

controlled through structural manipulation of UPP synthase of M. luteus

B-P 26, based on comparisons between structures of various cis-prenyl￾transferases. Replacements of Ala72, Phe73, and Trp78, which are located

in the proximity of the substrate binding site, with Leu ) as in Z,E-farnesyl

diphosphate (C15) synthase ) resulted in shorter ultimate products with

C20)35. Additional mutation of F223H resulted in even shorter products.

On the other hand, insertion of charged residues originating from long￾chain cis-prenyltransferases into helix-3, which participates in constitution

of the large hydrophobic cleft, resulted in lengthening of the ultimate prod￾uct chain length, leading to C60)75. These results helped us understand

reaction mechanisms of cis-prenyltransferase including regulation of the

ultimate prenyl chain-length.

Abbreviations

DedolPP, dehydrodolichyl diphosphate; E,E-FPP, E,E-farnesyl diphosphate; FARM, first aspartate-rich motif; GPP, geranyl diphosphate; IPP,

isopentenyl diphosphate; UPP, undecaprenyl diphosphate; Z,E-FPP, Z,E-farnesyl diphosphate; Z,E-DecPP, Z,E-mixed decaprenyl diphosphate.

FEBS Journal 273 (2006) 647–657 ª 2006 The Authors Journal compilation ª 2006 FEBS 647