White Rose University Consortium logo
University of Leeds logo University of Sheffield logo York University logo

Kinetic and biophysical approaches to the assembly of magnesium chelatase

Taurino, Luke (2016) Kinetic and biophysical approaches to the assembly of magnesium chelatase. PhD thesis, University of Sheffield.

[img] Text
Restricted until April 2022.

Request a copy


Magnesium chelatase catalyses the first committed step in chlorophyll biosynthesis, the insertion of Mg2+ into protoporphyrin-IX yielding magnesium protoporphyrin-IX; This enzyme stands at a branch-point in chlorophyll and heme biosynthesis. Mg2+ insertion is energetically unfavourable and is coupled with ATP hydrolysis. Magnesium chelatase from Synechocystis sp. PCC6803 contains three essential protein subunits ChlI, ChlD and ChlH. ChlI and ChlD belong to the AAA+ family of ATPases and form motor complexes that catalyse ATP hydrolysis which drives the Mg2+ insertion reaction occurring on ChlH. The number and arrangement of these protein subunits that represent the active chelatase are poorly understood. My work used a series of techniques to probe the complexes formed by the chelatase. Kinetic titrations show active complexes that catalyse either Mg2+ insertion or ATP hydrolysis occur at two widely different levels of subunits, either 1:1 levels or at much higher levels of ChlI over ChlD. ChlH influences these ChlID complexes, with high levels of ChlH appearing to cause a reorganisation of higher order ChlID complexes as well as causing a dramatic change in the ATPase activity of the system. An I6D6 complex has been demonstrated previously within the Rhodobacter capsulatus chelatase and a similar complex could be expected within the Synechocystis system. Sedimentation velocity analytical ultra centrifugation performed on the Synechocystis chelatase gives no evidence for this complex. A smaller complex is observed, favoured at high levels of ChlI, or in the presence of ChlH, this complex was proposed to represent an I5D complex or a similar arrangement. No ChlIDH complex was observed, although ChlH appears to influence the arrangement of smaller ChlID complexes. Modelling of the reaction cycle provides evidence for an isomerisation of ChlH preceding DIX substrate binding that may contribute to the observed lag phase prior to Mg2+ chelation.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Chemistry (Sheffield)
Depositing User: Mr Luke Taurino
Date Deposited: 09 Jun 2017 12:58
Last Modified: 09 Jun 2017 12:58
URI: http://etheses.whiterose.ac.uk/id/eprint/17524

Please use the 'Request a copy' link(s) above to request this thesis. This will be sent directly to someone who may authorise access.
You can contact us about this thesis. If you need to make a general enquiry, please see the Contact us page.

Actions (repository staff only: login required)