Pigment-free NADPH:protochlorophyllide oxidoreductase from Avena sativa L. Purification and substrate specificity

Eur J Biochem. 1999 Nov;265(3):862-74. doi: 10.1046/j.1432-1327.1999.00627.x.

Abstract

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) is the key enzyme for light-dependent chlorophyll biosynthesis. It accumulates in dark-grown plants as the ternary enzyme-substrate complex POR-protochlorophyllide a-NADPH. Here, we describe a simple procedure for purification of pigment-free POR from etioplasts of Avena sativa seedlings. The procedure implies differential solubilization with n-octyl-beta-D-glucoside and one chromatographic step with DEAE-cellulose. We show, using pigment and protein analysis, that etioplasts contain a one-to-one complex of POR and protochlorophyllide a. The preparation of 13 analogues of protochlorophyllide a is described. The analogues differ in the side chains of the macrocycle and in part contain zinc instead of the central magnesium. Six analogues with different side chains at rings A or B are active substrates, seven analogues with different side chains at rings D or E are not accepted as substrates by POR. The kinetics of the light-dependent reaction reveals three groups of substrate analogues with a fast, medium and slow reaction. To evaluate the kinetic data, the molar extinction coefficients in the reaction buffer had to be determined. At concentrations above 2 mole substrate/mole enzyme, inhibition was found for protochlorophyllide a and for the analogues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Avena / enzymology*
  • Chromatography, High Pressure Liquid
  • Kinetics
  • Oxidoreductases / chemistry
  • Oxidoreductases / isolation & purification*
  • Oxidoreductases / metabolism*
  • Oxidoreductases Acting on CH-CH Group Donors*
  • Photochemistry
  • Protochlorophyllide / analogs & derivatives
  • Protochlorophyllide / chemistry
  • Protochlorophyllide / metabolism
  • Spectrophotometry
  • Substrate Specificity

Substances

  • Protochlorophyllide
  • Oxidoreductases
  • Oxidoreductases Acting on CH-CH Group Donors
  • protochlorophyllide reductase