Award details

Mechanistic and structural studies on chorismate-utilising enzymes

Reference	BB/C513918/1
Principal Investigator / Supervisor	Professor Chris Abell
Co-Investigators / Co-Supervisors	Professor Sir Tom Blundell
Institution	University of Cambridge
Department	Chemistry
Funding type	Research
Value (£)	266,529
Status	Completed
Type	Research Grant
Start date	10/06/2005
End date	09/06/2008
Duration	36 months

Abstract

We propose to study four enzymes (PabB, EntC, Irp9 and TrpE) involved in making essential aromatic compounds including p-aminobenzoate, salicylate and anthranilate. The enzymes all use chorismate by convert it into four different products. We want to understand the mechanism of each of the enzymes. We will explore the hypothesis that they use a related mechanism to replace the C-4 hydroxyl of chorismate with a nucleophilic group at C-2, but at that point the mechanisms diverge. In particular Irp9 and TrpE cleave off the enol pyruvyl side chain and make aromatic products, whereas the other two do not. Sequence alignments and (some) structural comparisons show that the active sites of all four enzymes are very similar but provide a few clues to explain the different chemistries. Our research in this area is internationally competitive. We have determined the mode of action of irreversible inhibitor on PabB which provided novel insight into the role of Lys274 in the catalytic mechanism. Very recently we trapped the putative covalent intermediate in the PabB reaction and detected it by electrospray mass spectrometry. We will use 13C NMR spectroscopy and x-ray crystallography to determine the structure of the intermediate on PabB. We plan to use stopped experiments coupled with mass spectrometry to measure the rate of formation of the intermediate. The intermediate reacts with ammonia, which is generated from glutamine on an associated protein (PabA). This reaction procedes via a glutaminyl thioester on PabA that we can also detect by mass spectrometry. We plan to follow the two coupled reactions on the PabA:PabB complex simultaneously by mass spectrometry. The ammonia generated on PabA is thought to pass through a tunnel to the active site of PabB. We hope to get information about this tunnel by solving the crystal structure of the PabA:PabB complex. Our approach to addressing the question of how some enzymes cleave off the enol pyruvyl side chain and others do not, isto study two very closely-related enzymes, both (misleadingly) known as isochorismate synthases (ICSs). These are: EntC, a monofunctional ICS that converts chorismate into isochorismate; and Irp9, a bifunctional ICS that converts chorismate initially into isochorismate and then on to salicylate. Our work on Irp9 is world leading, we are the first group to overexpress a bifunctional ICS. We will make and characterise site-directed mutants of EntC and Irp9 to see how the changes affect the rate and kind of chemistry they catalyse. We will use x-ray crystallography to determine (for the first time) the structure of a monofunctional ICS (EntC) and a bifunctional ICS (Irp9). The structural differences should help explain why Irp9 cleaves off the side chain and EntC does not. Sequence alignments suggest that the ICSs will be structurally related to PabB and TrpE. The subtle structural differences will be of great interest in relation to the site of nucleophilic attack (C-2 v C4) and the nature of the nucleophile (ammonia v water). We will use in vitro molecular evolution to evolve one enzyme activity into another. The first study will involve generating variants of PabB which catalyse the TrpE reaction. This will require a change in the site of amination and introduction of the side chain lyase activity. The pabB gene will be subjected to error-prone PCR mutagenesis and or DNA shuffling, then introduced into a trpE deleted strain, which can be supplemented with o-aminobenzoate. We will try to understand the role of the putative tryptophan binding site in PabB, EntC, Irp9 and TrpE. Tryptophan clearly inhibits TrpE allosterically. Limited evidence suggests it is required for the structural integrity of PabB, while nothing is known about its role in regulation ICS.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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