Overexpression, single-step purification, and site-directed mutagenetic analysis of casbene synthase.
Casbene synthase is a diterpene cyclase isolated from castor bean (Ricinus communis L), which catalyzes the cyclization of geranylgeranyl diphosphate to form the phytoalexin casbene. We here report the overexpression of casbene synthase in Escherichia coli in 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 beta-d-thiogalactopyranoside at 20 degrees 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 in Km as well as a reduction in kcat in 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.
Huang
K
K
Chemistry Department, Texas A&M University, College Station, Texas 77843-3255, USA.
Huang
Q l
Ql
Scott
A I
AI
D000595:Amino Acid Sequence; D001483:Base Sequence; D002367:Ricinus communis / Q000201:enzymology / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C044955:casbene synthetase; D019761:Phosphorus-Oxygen Lyases