Purification of solanesyl-diphosphate synthase from Micrococcus luteus. A new class of prenyltransferase.
The activity of solanesyl-diphosphate synthase from Micrococcus luteus is stimulated by a high molecular mass fraction (HMF) which is separated from cell-free extracts of the same bacterium by DEAE-Toyopearl chromatography followed by Sephadex G-100 chromatography. By employing HMF in the assay procedure, solanesyl-diphosphate synthase was able to be purified to homogeneity and was found to be a homodimer with a monomeric molecular mass of 34 kDa. In contrast to hexaprenyl- and heptaprenyl-diphosphate synthases, which are composed of two easily dissociable components that are inactive unless combined, the homogeneously purified solanesyl-diphosphate synthase itself showed a catalytic activity, though weak, catalyzing the synthesis of both (all-E)-nonaprenyl-(solanesyl-) and (all-E)-octaprenyl diphosphate. HMF does not affect the stability of solanesyl-diphosphate synthase or Km values for isopentenyl diphosphate and farnesyl diphosphate, but it markedly increases Vmax values in a time-dependent manner. Several lines of evidence indicate that HMF contains a factor which binds to polyprenyl products and removes them out of the active site of enzyme to facilitate and maintain the turnover of catalysis.
Ohnuma
S
S
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
Koyama
T
T
Ogura
K
K
D019883:Alkyl and Aryl Transferases; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007700:Kinetics; D016982:Micrococcus luteus / Q000201:enzymology*; D008970:Molecular Weight; D014166:Transferases / Q000302:isolation & purification* / Q000378:metabolism
D014166:Transferases; D019883:Alkyl and Aryl Transferases; C022644:trans-octaprenyltranstransferase