Pentalenene synthase. Purification, molecular cloning, sequencing, and high-level expression in Escherichia coli of a terpenoid cyclase from Streptomyces UC5319.
Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.
Cane
D E
DE
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Sohng
J K
JK
Lamberson
C R
CR
Rudnicki
S M
SM
Wu
Z
Z
Lloyd
M D
MD
Oliver
J S
JS
Hubbard
B R
BR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D019753:Intramolecular Lyases; D007535:Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007535:Isomerases; D019753:Intramolecular Lyases; C048278:farnesylpyrophosphate cyclase