Protein design of geranyl diphosphate synthase. Structural features that define the product specificities of prenyltransferases.
Geranyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give a C(10) compound, geranyl diphosphate, which is a precursor of all monoterpenoids. However, the gene has not been isolated from any organisms. To examine the possibility that geranyl diphosphate synthase has evolved from a common ancestor of the prenyltransferase family and to predict the active site structure, we tried to convert Bacillus stearothermophilus farnesyl diphosphate synthase to geranyl diphosphate synthase, according to our previous findings. Several mutated farnesyl diphosphate synthases that have single amino acid substitutions before the first aspartate-rich motif were constructed. A mutated enzyme that has the replacement of serine by phenylalanine at the fourth position before the motif exclusively produced geranyl diphosphate when dimethylallyl diphosphate was used as the primer, and hardly accepted geranyl diphosphate as a primer, indicating that this mutation causes the conversion to geranyl diphosphate synthase. This result supports the idea that the product specificities of all members of the E-prenyltransferase family are mainly defined by a few structural features: the amino acids at the fourth position and the fifth position before the first aspartate-rich motif, and the insertion of two amino acids in the motif. This suggests that natural geranyl diphosphate synthases might have an active site structure similar to that of the mutated enzyme.
Narita
K
K
Department of Biochemistry and Engineering, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, 980-8579, Japan.
Ohnuma
S
S
Nishino
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D019753:Intramolecular Lyases; C044526:geranyl-diphosphate cyclase