A pathway where polyprenyl diphosphate elongates in prenyltransferase. Insight into a common mechanism of chain length determination of prenyltransferases.
Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an alpha-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996) J. Biol. Chem. 271, 18831-18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40 compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.
Department of Biochemistry and Engineering, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai 980-8579, Japan.
S
S
Ohnuma
K
K
Hirooka
N
N
Tsuruoka
M
M
Yano
C
C
Ohto
H
H
Nakane
T
T
Nishino
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D051231:Farnesyltranstransferase; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000378:metabolism*
D017931:DNA Primers; D009943:Organophosphorus Compounds; D019883:Alkyl and Aryl Transferases; D051231:Farnesyltranstransferase