Chlorophyll biosynthesis in barley: Studies on the cyclase and chlorophyll synthase steps

The well-known green color of plants is due to the chlorophyll molecules used for light harvesting
during photosynthesis, which takes place in chloroplasts. Biosynthesis of chlorophyll a is performed by
15 enzymatic steps and chlorophyll a can be interconverted to chlorophyll b via the chlorophyll cycle.
Barley is a valuable model organism for studying chloroplast development and chlorophyll biosynthesis
thanks to the availability of many chlorophyll mutants with abnormal pigment accumulation. Many of
these mutants are deficient in enzymes of chlorophyll biosynthesis. Using barley as a model system, I
have performed studies of two enzymatic steps in the chlorophyll biosynthetic pathway; the aerobic
magnesium-protoporphyrin IX monomethyl ester cyclase and the chlorophyll synthase. I have
identified the cyclase as a ferredoxin dependent enzyme and established the first in vitro assay using
recombinant cyclase enzyme. I have also utilized the barley viridis-k chlorophyll mutants, which have
impaired chlorophyll synthesis due to deficiency at the cyclase step. This revealed that a particular
ferredoxin isoform, FdC2, is likely the main in vivo electron donor to the cyclase. By developing a
pipeline for identification of genes deficient in barley mutants and applying this on barley xantha-j
mutants, I connected this locus to the chlorophyll synthase. An AlphaFold generated structural model
of the chlorophyll synthase suggested the active site to be subdivided into a prenyl pyrophosphate
tunnel, the catalytic cavity, and a tetrapyrrole-binding pocket. A deep multiple sequence alignment
contributed to the understanding of amino-acid residues. I suggest that binding of the isoprenoid
substrate is a prerequisite for stable maintenance of chlorophyll synthase in the plastid. I further
suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.