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Aerobic barley mg-protoporphyrin ix monomethyl ester cyclase is powered by electrons from ferredoxin

Stuart, David LU ; Sandström, Malin ; Youssef, Helmy M. LU ; Zakhrabekova, Shakhira LU ; Jensen, Poul Erik ; Bollivar, David W. LU and Hansson, Mats LU (2020) In Plants 9(9).
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... (More)

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.

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author
; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
AcsF, BchE, CHL27, Chlorophyll biosynthesis, CRD1, FNR, Hordeum vulgare, XanL, Xantha-l
in
Plants
volume
9
issue
9
article number
1157
pages
13 pages
publisher
Oxford University Press
external identifiers
  • scopus:85090689859
  • pmid:32911631
ISSN
2041-2851
DOI
10.3390/plants9091157
language
English
LU publication?
yes
id
35317372-b423-47c8-84b1-f2b1e2bde64c
date added to LUP
2020-09-30 14:04:03
date last changed
2024-05-01 16:57:59
@article{35317372-b423-47c8-84b1-f2b1e2bde64c,
  abstract     = {{<p>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.</p>}},
  author       = {{Stuart, David and Sandström, Malin and Youssef, Helmy M. and Zakhrabekova, Shakhira and Jensen, Poul Erik and Bollivar, David W. and Hansson, Mats}},
  issn         = {{2041-2851}},
  keywords     = {{AcsF; BchE; CHL27; Chlorophyll biosynthesis; CRD1; FNR; Hordeum vulgare; XanL; Xantha-l}},
  language     = {{eng}},
  number       = {{9}},
  publisher    = {{Oxford University Press}},
  series       = {{Plants}},
  title        = {{Aerobic barley mg-protoporphyrin ix monomethyl ester cyclase is powered by electrons from ferredoxin}},
  url          = {{http://dx.doi.org/10.3390/plants9091157}},
  doi          = {{10.3390/plants9091157}},
  volume       = {{9}},
  year         = {{2020}},
}