Record number :
1612885
Title of article :
Overexpression, Single-Step Purification, and Site-Directed Mutagenetic Analysis of Casbene Synthase
Author/Authors :
Huang، نويسنده , , Ke-xue and Huang، نويسنده , , Qiu-long and Scott، نويسنده , , A.I.، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 1998
Pages :
9
From page :
144
To page :
152
Abstract :
Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communisL), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene. We here report the overexpression of casbene synthase inEscherichia coliin soluble form using a thioredoxin fusion system. The amplified DNA by PCR carried on pCS7 was inserted into the expression vector pET32b(+) to form pCAS.2. The resulting transformants of pCAS.2/BL21(DE3) produced a thioredoxin casbene synthase fusion protein (20–30% of total soluble protein) when induced with isopropyl β-d-thiogalactopyranoside at 20°C. Recombinant casbene synthase was purified to homogeneity in a single step with a His-binding metal-affinity column. Casbene synthase has a conserved aspartate-rich region [amino acids 355–359 (DDTID)], one cysteine, and three histidines with several prenyl transferases and terpene cyclases. Seven mutants were constructed by site-directed mutagenesis. The importance of Asp 355 and Asp 356 for catalysis was established by an increase inKmas well as a reduction inkcatin the corresponding glutamate mutants. These results indicate that the first and the second aspartate are involved in catalysis, while the third aspartate and the conserved cysteine and histidine residues selected for mutagenesis appear not to be involved in catalysis.
Keywords :
casbene synthase , Geranylgeranyl diphosphate , site-directed mutagenesis
Journal title :
Archives of Biochemistry and Biophysics
Link To Document :