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Title: The aerobic cyclase involved in (bacterio)chlorophyll biosynthesis
Author: Chen, Guangyu
ISNI:       0000 0004 5993 2566
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Photosynthesis is essential for almost all life on Earth. Chlorophylls are essential for photosynthesis and are modified tetrapyrrole molecules containing a centrally chelated magneiusm ion and a unique isocyclic E ring. The formation of the E ring is catalysed by the magnesium-protoporphyrin IX monomethyl ester cyclase (the cyclase). Two fundamentally distinct types of the cyclase exist in photosynthetic organisms, utilising an oxygen atom from either water (the anaerobic cyclase) or molecular oxygen (the aerobic cyclase). The aerobic cyclase has remained an enigma for over 65 years and it was proposed to be a multi-subunit enzyme. The first subunit was identified in the purple bacterium Rubrivivax gelatinosus and designated as AcsF, which is the catalytic subunit and contains a di-iron binding motif. AcsF is conserved across all phototrophs that possess an aerobic cyclase. Ycf54 was identified as a possible second subunit in the cyanobacterium Synechocystis sp. PCC6803 and is conserved in all oxygenic phototrophs. Previous studies suggested that there are more, unknown subunits required for the aerobic cyclase. This thesis focuses on studying the subunit composition of aerobic cyclase with extensive genetic engineering conducted in several photosynthetic bacteria. Rhodobacter sphaeroides, one of the principal model organisms to study bacterial photosynthesis, was shown to harbour a functional aerobic cyclase. BciE was subsequently identified to be the second aerobic cyclase subunit in this organism. Complementation profiles in a Rubrivivax gelatinosus mutant lacking both the anaerobic and aerobic cyclases lead to the identification of three classes of aerobic cyclase as represented by the enzymes from Rhodobacter sphaeroides (AcsF + BciE), Rubrivivax gelatinosus (AcsF) and Synechocystis sp. PCC6803 (AcsF + Ycf54), respectively. The distribution of BciE and Ycf54 across phototrophs is well correlated with the evolutionary history of the AcsF proteins. A suppressor screen conducted with a Ycf54-lacking mutant of Synechocystis sp. PCC6803 did not reveal any additional subunit of aerobic cyclase. Likewise, transposon mutagenesis performed in a Rubrivivax gelatinosus mutant lacking the anaerobic cyclase did not uncover any new subunit of aerobic cyclase. The aerobic cyclase activity was demonstrated in vivo with an Escherichia coli strain expressing the Rubrivivax gelatinosus AcsF protein, providing conclusive evidence that no additional subunit is required for the aerobic cyclase. Finally, the core pathway of chlorophyll biosynthesis, from protoporphyrin IX to chlorophyllide a, was successfully constructed in Escherichia coli.
Supervisor: Hunter, Neil Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available