The University of Southampton
University of Southampton Institutional Repository

Structure and function of the alpha beta hydrolase MhpC

Structure and function of the alpha beta hydrolase MhpC
Structure and function of the alpha beta hydrolase MhpC
2-hydroxy-6 keto nona 2, 4 diene 1,9 dioic acid 5,6 hydrolase (MhpC) is a homodimeric enzyme belonging to the ?/? hydrolase fold family. The three catalytic residues comprise Ser110, Asp235 and His263. To study structure and catalytic mechanism of MhpC, Ser110 was replaced by cysteine and aspartic acid. A range of other mutants were also investigated. The S110C mutant enzyme showed 400 fold reduced catalytic efficiency compared with wild type enzyme, implying that it is a crucial residue in catalysis. The crystal structure of MhpC S110C demonstrated a water molecule interleaving between Cys110 and His263. Exposure of mutant enzyme crystals to substrate showed formation of a covalent complex with the product (hydroxypentadienoic acid) bound through C3 probably by a Michael adduct reaction with the Cys110 thiol. The enzyme-product adduct was confirmed by mass spectrometry. Interestingly, the intact enzyme-substrate complex was also observed by mass spectrometry. The S110D mutant enzyme was not active. The crystal structure of MhpC S110D showed that the active site residue His263 was slightly moved, forming a good hydrogen bond with D110, and many residues in the active site region (Trp264, Phe173 and Phe237) were rearranged. The carboxylate group may provide an interesting mimic of a gem-diol transition state. Serine 40 is close to the catalytic triad and S40C enzyme was 18 fold less active than wild type enzyme. The crystal structure of MhpC S40C revealed an interleaving water molecule between Ser110 and His263. The S40C/S110C double mutant succeeded to form a disulphide bridge redox-switch in the enzyme active site. To disrupt half-of-sites reactivity of MhpC a number of residues were mutated at the subunit interface of the dimeric enzyme. Radical changes in residue type led to mis-folding of MhpC. The more conservative N109G/H256N double mutant enzyme was correctly folded and dimeric although a charged substituent was no longer present in the hydrogen bonding network at the interface. Titration of charged interface residues showed monomeric MhpC by gel filtration at low pH but enzyme activity is low in these conditions.
Burakorn, Jiraporn
a0821cba-fb6c-4158-8112-877b5594a796
Burakorn, Jiraporn
a0821cba-fb6c-4158-8112-877b5594a796
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0

Burakorn, Jiraporn (2009) Structure and function of the alpha beta hydrolase MhpC. University of Southampton, School of Biological Sciences, Doctoral Thesis, 267pp.

Record type: Thesis (Doctoral)

Abstract

2-hydroxy-6 keto nona 2, 4 diene 1,9 dioic acid 5,6 hydrolase (MhpC) is a homodimeric enzyme belonging to the ?/? hydrolase fold family. The three catalytic residues comprise Ser110, Asp235 and His263. To study structure and catalytic mechanism of MhpC, Ser110 was replaced by cysteine and aspartic acid. A range of other mutants were also investigated. The S110C mutant enzyme showed 400 fold reduced catalytic efficiency compared with wild type enzyme, implying that it is a crucial residue in catalysis. The crystal structure of MhpC S110C demonstrated a water molecule interleaving between Cys110 and His263. Exposure of mutant enzyme crystals to substrate showed formation of a covalent complex with the product (hydroxypentadienoic acid) bound through C3 probably by a Michael adduct reaction with the Cys110 thiol. The enzyme-product adduct was confirmed by mass spectrometry. Interestingly, the intact enzyme-substrate complex was also observed by mass spectrometry. The S110D mutant enzyme was not active. The crystal structure of MhpC S110D showed that the active site residue His263 was slightly moved, forming a good hydrogen bond with D110, and many residues in the active site region (Trp264, Phe173 and Phe237) were rearranged. The carboxylate group may provide an interesting mimic of a gem-diol transition state. Serine 40 is close to the catalytic triad and S40C enzyme was 18 fold less active than wild type enzyme. The crystal structure of MhpC S40C revealed an interleaving water molecule between Ser110 and His263. The S40C/S110C double mutant succeeded to form a disulphide bridge redox-switch in the enzyme active site. To disrupt half-of-sites reactivity of MhpC a number of residues were mutated at the subunit interface of the dimeric enzyme. Radical changes in residue type led to mis-folding of MhpC. The more conservative N109G/H256N double mutant enzyme was correctly folded and dimeric although a charged substituent was no longer present in the hydrogen bonding network at the interface. Titration of charged interface residues showed monomeric MhpC by gel filtration at low pH but enzyme activity is low in these conditions.

PDF
jiraporn_thesis.pdf - Other
Restricted to Repository staff only
PDF
jiraporn_thesis_cover.pdf - Other
Restricted to Repository staff only

More information

Published date: May 2009
Organisations: University of Southampton

Identifiers

Local EPrints ID: 66709
URI: https://eprints.soton.ac.uk/id/eprint/66709
PURE UUID: 66496c0b-c2fa-4496-a488-71b4c1eb5c33

Catalogue record

Date deposited: 15 Mar 2010
Last modified: 19 Jul 2017 00:22

Export record

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of https://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×