Cloning and functional analysis of novel short-chain cis-prenyltransferases.
cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by sequential condensation of isopentenyl diphosphate with allylic diphosphate. cis-Prenyltransferases can be classified into three subgroups: short-, medium-, and long-chain cis-prenyltransferase, according to their product chain lengths. cis-Farnesyl diphosphate synthase from Mycobacterium tuberculosis has been the only example as short-chain cis-prenyltransferase so far characterized. In this study, we cloned the novel short-chain cis-prenyltransferases from three different bacteria, and characterized their enzymatic activities to compare and elucidate a common feature of the short-chain cis-prenyltransferases. Furthermore, we identified a specific isoleucine that is conserved in short-chain cis-prenyltransferases and located in close proximity of the omega-end of the geranyl diphosphate. Several site-directed mutants with respect to the isoleucine residue synthesized longer prenyl chain products and showed broader allylic substrate specificity. These results suggested that the isoleucine plays an important role in the substrate specificity and chain length determination mechanism of cis-prenyltransferase.
Ambo
Takanori
T
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
Noike
Motoyoshi
M
Kurokawa
Hirofumi
H
Koyama
Tanetoshi
T
D000192:Actinomycetales / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013379:Substrate Specificity; D014166:Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D007532:Isoleucine; D014166:Transferases; C106821:cis-prenyl transferase