Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin

David Stuart, Malim Sandstrom, Helmy M. Youssef, Shakira Zakhrabekova, Poul Erik Jensen, David Bollivar, Mats Hansson

Research output: Journal ArticleArticlepeer-review

Abstract

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis.
Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized
using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic
of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase
encoded by Xantha-l in barley ( Hordeum vulgare L.). The Xantha-l gene product (XanL) is a
membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound
components for its activity. XanL has so far been impossible to produce as an active recombinant protein
for in vitro assays, which is required for deeper biochemical and structural analyses. In the present
work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts.
Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited
assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and
FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL
required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained
with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates
that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic
electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis
under light conditions.
Original languageAmerican English
JournalPlants
Volume9
DOIs
StatePublished - Sep 8 2020

Keywords

  • acsF
  • bchE
  • CHL27
  • chlorophyll biosynthesis
  • CRD1
  • FNR
  • Hordeum vulgare
  • XanL
  • Xantha-l

Disciplines

  • Plant Sciences

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